Compounds and compositions including phosphorothioated oligodeoxynucleotide, and methods of use thereof

ABSTRACT

The present disclosure relates to a compound including a nucleic acid sequence conjugated to an anti-microRNA or a microRNA-mimic or a compound including a modified anti-microRNA sequence, compositions of such a compound, and method of treatment of a disease, and method of suppressing microRNA activity by the disclosed compound or composition.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/010,259 filed Sep. 2, 2020, allowed, which is a continuation of U.S.application Ser. No. 15/768,405 filed Apr. 13, 2018, issued as U.S. Pat.No. 10,801,026, which is a national stage application filed under 35U.S.C. § 371 of International Patent Application No. PCT/US2016/057143filed Oct. 14, 2016, which claims the benefit of and priority under 35U.S.C. § 119(e) to U.S. Application No. 62/242,189 filed Oct. 15, 2015,the entire contents of each of which are hereby expressly incorporatedby reference herein.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The contents of the XML file named 048440-588C02US SEQUENCE LISTINGST26.XML, created on Feb. 7, 2023, and is 84,164 bytes in size, arehereby incorporated by reference in their entireties.

BACKGROUND OF THE DISCLOSURE

Acute myeloid leukemia is characterized by accumulation of immaturemyeloid progenitor cells. Leukemogenesis results from deregulation ofoncogenes, tumor suppressors or transcription factors which controlmyeloid lineage differentiation, self-renewal and/or proliferation. Thepresent disclosure relates to compounds, compositions, and methods oftreating cancer, e.g., AML, CML and myelodysplastic syndrome, withanti-miRs and miRNA mimics that are stable and are suitable for systemicadministration against disseminated cancers.

BRIEF SUMMARY OF THE DISCLOSURE

In one aspect, provided herein is a compound including aphosphorothioated CpG oligodeoxynucleotide (CpG-ODN), conjugated to ananti-microRNA (anti-miR) or to a microRNA (miRNA)-mimic nucleic acidsequence (miRNA-mimic).

In another aspect, provided herein is a compound including ananti-microRNA (anti-miR) sequence, where the anti-miR sequence containsone or more phosphorothioate linkages and one or more chemicallymodified nucleotides.

In another aspect, provided herein is a pharmaceutical compositionincluding a pharmaceutically acceptable excipient and a compounddescribed herein.

In another aspect, provided herein is a method of treating a disease ina subject in need thereof. The method includes administering to thesubject an effective amount of a compound described herein or apharmaceutical composition described herein.

In another aspect, provided herein is a method of reducing the activityof microRNA in a cell. The method includes contacting the cell with aneffective amount of a compound described herein.

Other features and advantages of the disclosure will be apparent fromthe following detailed description and claims.

Unless noted to the contrary, all publications, references, patentsand/or patent applications reference herein are hereby incorporated byreference in their entirety for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic that shows the design of the chemically stabilizedCpG-anti-miR-126 RNA oligonucleotides. Asterix (*) indicatephosphorothioate linkages; and mN indicates a 2′OMe modified nucleotide.

FIGS. 2A-2D are flow cytometry histograms showing results of uptaketests in normal, cord blood, acute myeloid leukemia (AML), and chronicmyeloid leukemia (CML) CD34+ cells. Normal (NL), cord blood (CB), AMLand CML34+ cells were cultured with CpG-scramble RNA (500 nM) andCpG-miR126 inhibitor-Cy3 (500 nm) for 16 hours. FIG. 2A is a histogramshowing uptake in NL/CB cells with 600 CpG siRNA. FIG. 2B is a histogramshowing NL/CB cells with 500 CpG-siRNA. FIG. 2C is a histogram showinguptake in AML cells. FIG. 2D is a histogram showing uptake in CML cells.Uptake was measured by Cy3 expression in these cells by flow cytometry.

FIGS. 3A-3G are flow cytometry histograms showing results ofCpG-anti-miR-126 uptake in AML and CML cell lines. Cells were culturedwith miR126-inhibitor-Cy3 (Cy3-200 nM0, human CD45 (Ab-200 nM) orTransferrin (TF-200 nM)-conjugated nanoparticles (NP) containingantagomiR-126-Cy3, CpG-miR126 inhibitor-Cy3 (CpG-Cy3-200 nM and 500 nM)for 4 hours, then uptake was analyzed by measuring Cy3 expression inthese cells by flow cytometry. FIG. 3A is a histogram showing uptake inK562 cells. FIG. 3B is a histogram showing uptake in KG1A AML cells.FIG. 3C is a histogram showing uptake in MV4-11 AML cells. FIG. 3D is ahistogram showing uptake in Molm13 AML cells. FIG. 3E is a histogramshowing uptake in NB4 AML cells.

FIG. 3F is a histogram showing uptake in OCI AML cells. FIG. 3G is ahistogram showing uptake in HL60 AML cells.

FIGS. 4A-4G are bar graphs showing miRNA 126 expression in AML and CMLcell lines. Cells were cultured with miR126-inhibitor-Cy3 (Cy3-200 nM0,human CD45 (Ab-200 nM) or Transferrin (TF-200 nM)-conjugated NPcontaining antagomiR-126-Cy3, CpG-miR126 inhibitor-Cy3 (CpG-Cy3-200 nMand 500 nM) for 24 hours, then miR126 and RNU44 (control) expression inthese cells was analyzed by q-RT PCR. The miR126 expression levels werenormalized to RNU44 and calculated by using the comparative 2^(−ΔΔCt)method. FIG. 4A is a bar graph showing miRNA 126 expression in the K562CML cell line. FIG. 4B is a bar graph showing miRNA 126 expression inthe KG1A AML cell line. FIG. 4C is a bar graph showing miRNA 126expression in the MV4-11 AML cell line. FIG. 4D is a bar graph showingthe miRNA 126 expression in the Molm13 AML cell line. FIG. 4E is a bargraph showing the miRNA 126 expression in the NB4 AML cell line.

FIG. 4F is a bar graph showing the miRNA 126 expression in the OCI AMLcell line. FIG. 4G is a bar graph showing the miRNA 126 expression inthe HL60 AML cell line.

FIG. 5 is a bar graph showing the expression of miRNA 126 in NL/CB, AMLand CML CD34+ cells. Normal, cord blood, AML and CML CD34+ cells werecultured with CpG-scramble RNA (500 nM) and CpG-miR 126 inhibitor-Cy3(500 nM) for 24 hours. miR126 and RNU44 (control) expression wasanalyzed by q-RT-PCR. The miR126 expression levels were normalized toRNU44 and calculated by using the comparative 2^(−ΔΔCt) method.

FIG. 6 is a bar graph showing higher expression of miR126 in CMLCD34+CD38− primitive progenitors compared to CD34+CD38− committedprogenitors. CML CD34+, CD34+CD38+ committed and CD34+CD38− primitiveprogenitors were sorted and then miR126 and RNU44 (ctrl) expression inthese cells were analyzed by Q-RT-PCR. The miR126 expression levels werenormalized to RNU44 and calculated by using the comparative 2-ΔΔCtmethod.

FIG. 7 is a bar graph showing that miR126 expression was significantlyreduced in CML CD34+CD38+ committed and CD34+CD38− primitive cellstreated with CpG-mirR126 inhibitor. CML CD34+CD38+ committed andCD34+CD38− primitive progenitors were cultured with CpG-scramble RNA(500 nM) or CpG-miR126 inhibitor (500 nM) for 36 hours and then miR126and RNU44 (control) expression in these cells were analyzed by q-RT-PCR.The miR126 expression levels were normalized to RNU44 and calculated byusing the comparative 2^(−ΔΔCt) method.

FIG. 8 is a series of plots showing increased apoptosis of CML CD34+,CD34+CD38+ committed and CD34+CD38− primitive progenitors treated withCpG-miR126 inhibitor and nilotinib (NIL). CML CD34+, CD34+CD38+committed and CD34+CD38− primitive progenitors were cultured withCpG-scramble RNA (500 nM), CpG-miR126 inhibitor (500 nM), CpG-scrambleRNA (500 nM)+nilotinib (504), and CpG-miR126 inhibitor (500 nM)+NIL(504) for 72 hours and then apoptosis was analyzed by Annexin V/DAPIstaining.

FIGS. 9A-9D are a series of cell sorting plots showing increased cellcycle in CD34+CD38− cells after knockdown of miR-12. Normal (NL; FIGS.9A-9B) and CML human CD34+CD38− cells (FIGS. 9C-9D) were sorted and thencultured with 500 nm CpG-miR126 inh-Cy3 or CpG-SCR control for 72 hours,and then cell cycling was analyzed by EDU/DAPi staining. Increased cellcycling was seen in human CD34+CD38− cells after knockdown of miR-126(FIG. 9D).

FIGS. 10A-10D are a series of cell sorting plots showing increased cellcycling in normal (NL) and CML LTHSC after knockdown of miR-126. Normal(NL) and CML murine LTHSC (Lin-Sca-1+Kit+Fit3−CD150+CD48−cells) weresorted and then treated with 500 nm CpG-miR126 inhibitor or CpG-SCR for72 hours, then cell cycling was analyzed by EDU/DAPi staining. Increasedcell cycling was seen in NL and CML LTHSC after knockdown of miR-126(FIG. 10D).

FIGS. 11A-11B are bar graphs of cell sorting experiment showingsignificantly increased apoptosis and significant reduction of cellgrowth of LSC compared with SCR+NIL. Mouse CML leukemia stem cells (LSC,Lin-Sca-1+c-kit+Fit3-CD150+CD48−) were sorted and treated withCpG-anti-miR-126 inhibitor or CpG-SCR (500 nM) for 48 hours, thenfurthered treated with miR126 inhibitor+NIL or SCR+NIL for 72 hours,then apoptosis and cell growth were measured. CpG-anti-miR126inhibitor+NIL resulted in significantly increased apoptosis (FIG. 11A)and significant reduction of cell growth (FIG. 11B) of LSC compared withSCR+NIL.

FIG. 12A is a scatter plot and FIGS. 12B-12C are bar graphs showingsignificantly increased cell cycling and apoptosis compared withSCR+Ara-c+Doxo, resulting in reduction of cell growth. AML CD34+ cellswere cultured with CpG-anti-miR-126 or CpG-SCR (500 nM) for 48 hours,then furthered cultured with miR126 inh+Ara-c+Doxo or SCR+A+D for 72hours, then cell cycling, apoptosis (FIG. 12B) and cell growth (FIG.12C) were analyzed. CpG-anti-miR-126 inhibitor combined with Ara-c andDoxo significantly increased cell cycling and apoptosis compared withSCR+Ara-c+Doxo, resulting in reduction of cell growth.

FIGS. 13A-13D are scatter plots showing reduced spleen weight (FIG. 13B)and reduced CML cells observed in the bone marrow (BM) (FIG. 13C) andspleen (FIG. 13D) of the mice treated with NIL+miR126 inhibitor comparedwith the mice treated with NIL+SCR. SCLtTA/BCR-ABL mice were treatedwith CpG-miR-126 inhibitor (5 mg/kg, every other day, iv injection), SCR(5 mg/kg, every other day, iv injection), NIL(50 mg/kg, daily bygarage)+SCR, NIL+miR-126 inhibitor for 3 weeks and then the remainingCML cells in the PB (FIG. 13A), BM (FIG. 13C) and spleen (FIG. 13D) wereanalyzed. Reduced CML white blood cells in the PB of the mice treatedwith the NIL+miR126 inhibitor were seen (FIG. 13A), compared with themice treated with NIL+SCR. Reduced spleen weight (FIG. 13B) and reducedCML cells were observed in the BM (FIG. 13C) and spleen (FIG. 13D) ofthe mice treated with NIL+miR126 inhibitor compared with the micetreated with NIL+SCR.

FIGS. 14A-14D are scatter plots showing reduced CML LSK and LSC observedin the bone marrow (BM) (FIGS. 14A, 14C) and spleen (FIGS. 14B, 14D) ofthe mice treated with NIL+miR126 inhibitor compared with the micetreated with NIL+SCR. SCLtTA/BCR-ABL mice were treated with CpG-miR-126inhibitor, SCR, NIL+SCR, NIL+miR-126 inhibitor for 3 weeks and then theremaining CML LSK primitive cells and LSC in the BM and spleen wereanalyzed. Reduced CML LSK and LSC were observed in the BM (FIGS. 14A,14C) and spleen (FIGS. 14B, 14D) of the mice treated with NIL+miR126inhibitor compared with the mice treated with NIL+SCR.

FIG. 15 . Stability of CpG-anti-miRs. Half-life of chemically modifiedCpG-anti-miRs in 50% human serum. CpG-anti-miRs were incubated in humanserum at the concentration of 50% at 37° C. for up to 7 days. Thesamples were then resolved on 7.5 M Urea/20% PAGE gel and stained usingethidium bromide. The representative gel for CpG-anti-miR155 is shown.Graph showing quantification of band intensities combined from 3independent experiments. The estimated half-life is as indicated.

FIGS. 16A-16B. Cell-selective uptake of CpG-anti-miR146a. FIGS. 16A-16B)Dose and time dependent internalization of CpG-antimiR146a by targetimmune and leukemic cells without any transfection reagents.CpG-anti-miR146a was Cy3-labeled to detect the intracellular uptake bytarget cells using flow cytometry. FIG. 16A) Human immune cells wereincubated with indicated concentrations of CpG-anti-miR146acY3 for 1 hr.The uptake by CD14+ monocytes, CD1c+ mDCs, CD3+ T cells and CD19+B cellswas measured using flow cytometry. FIG. 16B) Cultured human AML cellsKG1a, MOLM13, and MOLM14 cells rapidly internalize CpG-anti-miR146acoeven a low concentrations.

FIGS. 17A-17F. Inhibitory effects of CpG-anti-miR155. FIGS. 17A-17F)CpG-anti-miR155 treatment reduces miR-155 expression in human and mousemyeloid cells. Mouse RAW264.7 (macrophages) (FIG. 17A) and DC2.4(dendritic cells) (FIG. 17B) as well as human MV4-11 AML cells (FIG.17C) were incubated with 100 nM CpG-anti-miR155 or CpG-scrambled RNA(negative control) for 18 h and then treated with 1 vg/ml LPS for 4 h.KG1a (FIG. 17D), MOLM13 (FIG. 17E), and MOLM14 (FIG. 17F) cells wereincubated with 100 nM CpG-anti-miR155 or CpG-scrambled RNA for 18 h.Mature miR-155 expression was measured using TAQMAN® qPCR assay andnormalized to snoRNA234 levels. miR-155 expression level in untreatedsamples was set as 1.0. Data are shown as means±SEM (n=3). *P<0.05.

FIGS. 18A-18F. Inhibitory effects of CpG-anti-miR125b. FIGS. 18A-18F)CpG-anti-miR125b treatment reduces miR-125b expression in various cellmodels. RAW264.7 (FIG. 18A), DC2.4 (FIG. 18B), and MV4-11 (FIG. 18C)cells were incubated with 100 nM CpG-anti-miR125b or CpG-scrambled RNA(negative control) for 18 h and then treated with 1 vg/mILPS for 4 h.Human AML cells-KG1a (FIG. 18D), MOLM13 (FIG. 18E), and MOLM14 (FIG.18F)—were incubated with 100 nM CpG-anti-miR125b or controlCpG-scrambled RNA for 18 h. Mature miR-125b expression was measuredusing TAQMAN® qPCR assay and normalized to snoRNA234 levels. miR-125bexpression level in untreated samples was set as 1.0. Data are shown asmeans±SEM (n=3). *P<0.05.

FIGS. 19A-19H. Inhibitory effects of CpG-anti-miR146a. FIGS. 19A-19F)CpG-anti-miR146a treatment reduces miR-146a expression in human andmouse myeloid cells. Mouse RAW264.7 (macrophages) (FIG. 19A) and DC2.4(dendritic cells) (FIG. 19B) as well as human MV4-11 (FIG. 19C) and KG1aAML (FIG. 19D) cells were incubated with 100 nM CpG-anti-miR146a orCpG-scrambled RNA (negative control) for 18 h and then treated with 1vg/ml LPS for 4 h. KG1a, MOLM13 (FIG. 19E), and MOLM14 (FIG. 19F) cellswere incubated with 100 nM CpG-anti-miR146a or CpG-scrambled RNA for 18h. FIGS. 19G-19H) Mouse CMM AML cells (FIG. 19G) and A20 lymphoma cells(FIG. 19H) were incubated with 100 nM CpG-miR146a mimic for 18 h. MaturemiR-146a expression was measured using TAQMAN® qPCR assay and normalizedto snoRNA234 levels. miR-146a expression level in untreated samples wasset as 1.0. Data are shown as means±SEM (n=3). *P<0.05.

FIGS. 20A-20D. Effects of CpG-anti-miRNAs on downstream targets. FIGS.20A-20C: CpG-anti-miRs regulate downstream targets of miR155, miR125b,and miR146a. Mouse RAW264.7 or human MV4-11 cells were incubated with250 nM or 500 nM of CpG-anti-miR155, CpG-anti-miR125b, orCpG-anti-miR146a, or 500 nM of CpG-scramble for 48 h, then the celllysates were collected and electrophoresed and immunoblotted byantibodies against SHIP1 (miR155 target) (FIG. 20A), IRF4 (miR125btarget) (FIG. 20B), or IRAK1 (miR146a target) (FIG. 20C). The bandintensities were normalized against (3-actin and quantified. Foldinduction over the control protein levels are indicated below the blot.FIG. 20D) MV4-11 cells were incubated with 500 nM of CpG-anti-miR155,CpG-antimiR125b, CpG-anti-miR146a, or CpG-scramble for 24 h, then celllysates were collected and electrophoresed and immunoblotted to detectactivated caspase 3 indicating induction of apoptosis.

FIGS. 21A-21H. Comparing the inhibition effects of CpG-anti-miR andGpC-anti-miR. FIGS. 21A-21D: CpG-anti-miR155, GpC-anti-miR155,CpG-anti-miR146a, and GpC-anti-miR146a treatment reduces miR155 ormiR-146a expression in RAW264.7 (FIGS. 21A, 21C) and A20 cells (FIGS.21B, 21D). The cells were incubated with 100 nM CpG-anti-miRs orGpC-anti-miRs for 18 hrs. FIGS. 21E-21H) CpG-anti-miRs and GpC-anti-miRstreatment regulates downstream targets of miR155 and miR146a. RAW264.7(FIGS. 21E, 21G) or A20 cells (FIGS. 21F, 21H) were incubated with 500nM of CpG-anti-miR155, GpC-anti-miR155, or CpG-anti-miR146a,GpC-anti-miR146a for 48 hrs, then the cell lysates were collected andimmunoblotted using antibodies against SHIP1 (miR155 target) or IRAK1(miR146a target)

FIGS. 22A-22E. CpG-miR146a mimic attenuates LPS induced inflammatorysignaling. FIGS. 22A-22B) CpG-miR146a mimic increases miR-146aexpression in cultured CMM leukemia (FIG. 22A) and A20 lymphoma cells(FIG. 22B). Cells were incubated with 100 nM CpG-miR146a mimic for 18 h.FIG. 22C) CpG-miR146a mimic inhibits IRAK1 expression, a downstreamtarget of miR146a. A20 cells were incubated with 500 nM of CpG-miR146amimic or LPS (used as a positive control) for 48 h, then the celllysates were collected and immunoblotted using IRAK1-specificantibodies. FIGS. 22D-22E) RAW-Blue cells, expressing NF-KB-responsivereporter gene, were treated with 500 nM of CpG-miR146a mimic for 24 hand then with 1 pg/ml LPS for another 24 h. Culture medium was collectedand analyzed for NF-KB activity using the Quanti-Blue assay kit (FIG.22D) for IL-6 levels in media using ELISA (FIG. 22E).

FIGS. 23A-23K. Effective in vitro and in vivo uptake and gene silencingeffects of the CpG-miR-126 inhibitor. Uptake test measured by flowcytometric analysis at 4 hours (FIG. 23A) and 24 hours (FIG. 23B) afteraddition of CpG-miR-126 inhibitor-Cy3 (CpG), Ab-NPs (Ab-NP) or TF-NPs(TF-NP) containing miR-126 inhibitor-Cy3, or naked miR-126 inhibitor-Cy3(labeled as Control in FIGS. 23A and B) in K562 cells. The experimentwas replicated twice. miR-126 expression in K562 was measured byQ-RT-PCR at 24 hours (n=3) (FIG. 23C). Uptake in HUVEC (FIG. 23D), humannormal (FIG. 23E) and CML (FIG. 23F) CD34⁺CD38⁻ cells at 4 hours afteraddition of CpG-miR-126 inhibitor-Cy3 (500 nM) was measured by flowcytometry. miR-126 expression in HUVEC (FIG. 23G), normal (FIG. 23H) andCML (FIG. 23I) CD34⁺CD38⁻ cells treated with CpG-miR-126 inhibitor (500nM) for 24 hours is shown (n=4). One of the two cell cycling experimentsin normal (FIG. 23J) and CML (FIG. 23K) CD34⁺CD38⁻ cells treated withCpG-miR-126 inhibitor (500 nM) by EDU staining is shown. Abbreviations:ab-NPs (CD45 antibody conjugated nanoparticles); TF-NPs (transferrin(TF)-conjugated nanoparticles).

FIGS. 24A-24I. Knockdown of miR-126 by CpG-miR-126 inhibitor enhanceselimination of mouse CML LSC in combination with NIL in vivo. BM cellsfrom SCL-tTA/BCR-ABL mice (CD45.2) were transplanted into congenic B6mice (CD45.1, n=40) to generate a cohort of mice with CML-like disease.Following confirmation of CML development at 4 weeks aftertransplantation, mice were randomly divided into 4 groups (n=10 each)and treated with CpG-miR-126 inhibitor (5 mg/kg i.v.4 times a week),CpG-scrRNA (5 mg/kg, i.v. 4 times a week), CpG-miR-126 inhibitor plusNIL (50 mg/kg, daily by gavage), and CpG-scrRNA plus NIL for 3 weeks.Percentage of donor CML cells in peripheral blood (PB) (FIG. 24A),spleen (FIG. 24B) and bone marrow (BM) (FIG. 24C), numbers of donor CMLLSK in spleen (FIG. 24D) and BM (FIG. 24E), and numbers of donor CMLlong term hematopoietic stem cells (LTHSC) in spleen (FIG. 24F) and BM(FIG. 24G) after 3 weeks' treatment were measured. Another cohort ofmice was treated for 3 weeks and then followed for survival studiesafter 3 weeks of treatment (n=10 in each group) (FIG. 24H). BM cells(CD45.2) from treated leukemic mice (3 weeks) were pooled, and 4×10⁶,2×10⁶, 1×10⁶, and 5×10⁵ cells/mouse were transplanted into secondarycongenic CD45.1 recipient mice irradiated at 900cGy (n=6mice/dose/condition×4 doses×4 conditions=96 mice). The recipient micewere monitored for 16 weeks for CML cell engraftment in blood andleukemia development by WBC count. Frequency of LIC was quantified usingL-Calc software (FIG. 24I). Abbreviations: NIL (Nilotinib); PB(peripheral blood); BM (bone marrow); LTHSC (long term hematopoieticstem cells); LIC (leukemia-initiating cells); LSK (lineage: Sca-1+c-kit+cells).

FIGS. 25A-25O. Effective in vitro and in vivo uptake and gene silencingeffects of the CpG-miR-126 inhibitor. Murine CML BM, LTHSC and EC cellswere treated with CpG-miR-126 inhibitor-Cy3 (500 nM) for 4 hours andthen Cy3+ cells were detected by flow cytometry (FIG. 25A). The cellswere also collected at 24 hours and miR-126 expression was determined byQ-RT-PCR (FIG. 25B). Cell cycling was measured by EDU staining at 72hours after addition of CpG-miR-126 inhibitor in CIVIL BM LTHSC. One ofthe two representative plots is shown in (FIG. 25C). CML mice weretreated with CpG-miR-126 inhibitor-Cy3 with one dose (5 mg/kg, ivinjection) and Cy3 uptake in BM, LTHSC and EC was measured at 16 hoursafter treatment by flow cytometry (FIG. 25D). Normal and CML mice werealso treated with CpG-miR-126 inhibitor (5 mg/kg/day, iv, daily) for 3days and BM, LTHSC and EC from femurs were sorted and miR-126 expressionwas determined by Q-RT-PCR (FIGS. 25E-25F). Wild type B6 mice weretreated with CpG-scrRNA (scrRNA) or CpG-miR-126 inhibitor (Inhibitor) (5mg/kg/day, iv injection) for 3 weeks and BM cells were collected andanalyzed. Red cell (RBC, FIG. 25G), WBC (FIG. 25H), PLT (FIG. 25I), BMmononuclear cell (FIG. 25J), LTHSC (FIG. 25K) and EC (FIG. 25L) numbersare shown. BM cells (CD45.2) from the treated normal mice weretransplanted into CD45.1 congenic recipient mice and the donor cellengraftment in blood (FIG. 25M) and in BM and spleen at 16 weeks (FIG.25N) and the donor LTHSC number in BM at 16 weeks (FIG. 25O) wasmonitored. Results shown represent mean±SEM. *p<0.05, **p<0.01,***p<0.001. Abbreviations: EC (endothelial cells); PLT (platelet).

FIGS. 26A-26E. Effective knockdown of miR-126 with miR-126 inhibitorsconjugated with CpG, GpC and PS, and effective over-expression withmiR-126 mimics in K562 and MV4-11 cells. K562 and MV4-11 cells weretreated with miR-126 inhibitors conjugated with CpG, GpC, PS (FIGS.26A-26B) or miR-126 mimics (615, 616 and 617) (FIGS. 26C-26D) (500 nM)for 24 hours, and miR-126 expression was measured in these cells. Weshowed here that CpG motif can be omitted in the targeting ODN sequence.GpC and completely PS-modified oligo also succeeds in blocking miR126.Incubation with miR-126 mimics, especially GM617, significantlyincreased miR-126 expression in K562 and MV4-11 cells. Just likeCpG-miR-126 inhibitor which is very effective in reducing miR-126 incells, we also designed miR-126 mimics, which are very effective inincreasing miR-126 levels in cells without using any transductionreagents. FIG. 26E: Tabulation of sequence set forth herein.

DETAILED DESCRIPTION OF THE DISCLOSURE

Provided herein, inter alia, a compound including a phosphorothioatedCpG oligodeoxynucleotide (CpG-ODN), conjugated to an anti-microRNA(anti-miR) or to a microRNA (miRNA)-mimic nucleic acid sequence(miRNA-mimic) or a compound including an anti-microRNA (anti-miR)sequence, where the anti-miR sequence contains one or morephosphorothioate linkages and one or more chemically modifiednucleotides. In embodiments, the compound described herein promotes theinternalization of anti-miRs and/or miRNA-mimic. In embodiments, themodifications/conjugates used herein facilitate the leaving of thecompounds described herein from endosomes. In embodiments, themodifications/conjugates used herein stabilize the anti-miRs and/ormiRNA-mimics used herein.

Definitions

The following definitions are included for the purpose of understandingthe present subject matter and for constructing the appended patentclaims. Abbreviations used herein have their conventional meaning withinthe chemical and biological arts.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Singleton et al., DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York,NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, ColdSprings Harbor Press (Cold Springs Harbor, N Y 1989). Any methods,devices and materials similar or equivalent to those described hereincan be used in the practice of this disclosure. The followingdefinitions are provided to facilitate understanding of certain termsused frequently herein and are not meant to limit the scope of thepresent disclosure.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single-, double- or multiple-stranded form,or complements thereof. The term “polynucleotide” refers to a linearsequence of nucleotides. The term “nucleotide” typically refers to asingle unit of a polynucleotide, i.e., a monomer. Nucleotides can beribonucleotides, deoxyribonucleotides, or modified versions thereof.Examples of polynucleotides contemplated herein include single anddouble stranded DNA, single and double stranded RNA (including siRNA),and hybrid molecules having mixtures of single and double stranded DNAand RNA. Nucleic acids can be linear or branched. For example, nucleicacids can be a linear chain of nucleotides or the nucleic acids can bebranched, e.g., such that the nucleic acids comprise one or more arms orbranches of nucleotides. Optionally, the branched nucleic acids arerepetitively branched to form higher ordered structures such asdendrimers and the like.

Nucleic acids, including nucleic acids with a phosphothioate backbonecan include one or more reactive moieties. As used herein, the termreactive moiety includes any group capable of reacting with anothermolecule, e.g., a nucleic acid or polypeptide through covalent,noncovalent or other interactions. By way of example, the nucleic acidcan include an amino acid reactive moiety that reacts with an amino acidon a protein or polypeptide through a covalent, non-covalent or otherinteraction.

The terms also encompass nucleic acids containing known nucleotideanalogs or modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, which have similarbinding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphodiester derivativesincluding, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate(also known as phosphothioate), phosphorodithioate, phosphonocarboxylicacids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformicacid,methyl phosphonate, boron phosphonate, or O-methylphosphoroamiditelinkages (see Eckstein, Oligonucleotides and Analogues: A PracticalApproach, Oxford University Press); and peptide nucleic acid backbonesand linkages. Other analog nucleic acids include those with positivebackbones; non-ionic backbones, modified sugars, and non-ribosebackbones (e.g. phosphorodiamidate morpholino oligos or locked nucleicacids (LNA)), including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Sanghui & Cook, eds. Nucleic acidscontaining one or more carbocyclic sugars are also included within onedefinition of nucleic acids. Modifications of the ribose-phosphatebackbone may be done for a variety of reasons, e.g., to increase thestability and half-life of such molecules in physiological environmentsor as probes on a biochip. Mixtures of naturally occurring nucleic acidsand analogs can be made; alternatively, mixtures of different nucleicacid analogs, and mixtures of naturally occurring nucleic acids andanalogs may be made.

As may be used herein, the terms “nucleic acid,” “nucleic acidmolecule,” “nucleic acid oligomer,” “oligonucleotide,” “nucleic acidsequence,” “nucleic acid fragment” and “polynucleotide” are usedinterchangeably and are intended to include, but are not limited to, apolymeric form of nucleotides covalently linked together that may havevarious lengths, either deoxyribonucleotides or ribonucleotides, oranalogs, derivatives or modifications thereof. Different polynucleotidesmay have different three-dimensional structures, and may perform variousfunctions, known or unknown. Non-limiting examples of polynucleotidesinclude a gene, a gene fragment, an exon, an intron, intergenic DNA(including, without limitation, heterochromatic DNA), messenger RNA(mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinantpolynucleotide, a branched polynucleotide, a plasmid, a vector, isolatedDNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, anda primer. Polynucleotides useful in the methods of the invention maycomprise natural nucleic acid sequences and variants thereof, artificialnucleic acid sequences, or a combination of such sequences.

A polynucleotide is typically composed of a specific sequence of fournucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine(T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus,the term “polynucleotide sequence” is the alphabetical representation ofa polynucleotide molecule; alternatively, the term may be applied to thepolynucleotide molecule itself. This alphabetical representation can beinput into databases in a computer having a central processing unit andused for bioinformatics applications such as functional genomics andhomology searching. Polynucleotides may optionally include one or morenon-standard nucleotide(s), nucleotide analog(s) and/or modifiednucleotides.

Unless indicated otherwise, the following annotations are used in thenucleic acid sequences disclosed herein: *=phosphorothioate linkage;xxxxx=any linker described herein and in embodiments xxxxx may be=—(CH₂)_(n)—PO₄—[(CH₂)_(n)—PO₄]_(z)—(CH₂)_(n)) bonded to phosphategroups at both ends except at the termini where terminal phosphates areoptionally added and 5′x has an OH terminus and 3′ x has a —C⁶—NH₂bonded to the final phosphate group, other linkages are phosphodiester;mN indicates a 2′OMe modified nucleotide; fN indicates a 2′fluoromodified nucleotide; and rN indicates a ribonucleotide.

As used herein, the term “anti-microRNA (anti-miR)” or “anti-microRNA(anti-miR) nucleic acid sequence” is used according to its plain andordinary meaning and refers to RNA that is capable of suppressing orreducing expression and/or activity of a target microRNA. Inembodiments, the anti-miR oligomer may be a single stranded oligomer of20-30 bases. In embodiments, the anti-miR oligomer may be a doublestranded oligomer of 20-30 bases. In embodiments, the anti-miR oligomermay be partially double stranded, with single stranded overhangs. Inembodiments, the oligomer may have a 2′chemical modification. Inembodiments, the oligomer may have serum stability-enhancing chemicalmodification, e.g., a phosphothioate internucleotide linkage, a2′-O-methyl ribonucleotide, a 2′-deoxy-2′fluoro ribonucleotide, a2′-deoxy ribonucleotide, a universal base nucleotide, a 5-C methylnucleotide, an inverted deoxybasic residue incorporation, or a lockednucleic acid. In embodiments, an anti-miR sequence hybridizes to thecorresponding miR sequence. Full complementarity is not necessarilyrequired, provided there is sufficient complementarity to causehybridization. In some embodiments, the degree of complementaritybetween an anti-miR sequence and its corresponding miR sequence, whenoptimally aligned using a suitable alignment algorithm, is about or morethan about 50%, 60%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%. Optimal alignment may be determined with the useof any suitable algorithm for aligning sequences, non-limiting exampleof which include the Smith-Waterman algorithm, the Needleman-Wunschalgorithm, algorithms based on the Burrows-Wheeler Transform (e.g. theBurrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign(Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP(available at soap.genomics.org.cn), and Maq (available atmaq.sourceforge.net). In embodiments, the anti-miR sequence has at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity withthe perfectly complementary sequence of the target miR sequence.

A “microRNA,” “microRNA nucleic acid sequence,” “miR,” “miRNA” as usedherein, refers to a nucleic acid that functions in RNA silencing andpost-transcriptional regulation of gene expression. The term includesall forms of a miRNA, such as the pri-, pre-, and mature forms of themiRNA. In embodiments, microRNAs (miRNAs) are short (20-24 nt)non-coding RNAs that are involved in post-transcriptional regulation ofgene expression in multicellular organisms by affecting both thestability and translation of mRNAs. miRNAs are transcribed by RNApolymerase II as part of capped and polyadenylated primary transcripts(pri-miRNAs) that can be either protein-coding or non-coding. Theprimary transcript is cleaved by the Drosha ribonuclease III enzyme toproduce an approximately 70-nt stem-loop precursor miRNA (pre-miRNA),which is further cleaved by the cytoplasmic Dicer ribonuclease togenerate the mature miRNA and antisense miRNA star (miRNA*) products.The mature miRNA is incorporated into a RNA-induced silencing complex(RISC), which recognizes target mRNAs through imperfect base pairingwith the miRNA and most commonly results in translational inhibition ordestabilization of the target mRNA. In embodiments, a miRNA nucleic acidsequence described herein is about 10 to 80 nucleotides (e.g., 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 nucleotides)in length. In embodiments, a miRNA nucleic acid sequence describedherein is about 15 to 50 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides) in length. Inembodiments, a miRNA nucleic acid sequence described herein is about 18to 25 nucleotides (e.g., 18, 19, 20, 21, 22, 23, 24, 25 nucleotides) inlength.

As used herein, the term “miR126” or “miR142 nucleic acid sequence”includes all forms of miR126 including the pri-, pre-, and mature formsof miR126, as well as variants, homologues, modifications, andderivatives thereof (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to the native miR126). Inembodiments, the variants or homologues or derivatives have at least50%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity across the whole sequence or a portion of the sequence(e.g. a 10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, or 80continuous nucleotides portion) compared to a naturally occurring form.In embodiments, the miR126 is the miRNA as identified by NCBI ReferenceSequence: NR 029695.1 or sequence: cgctggcgac gggacattat tacttttggtacgcgctgtg acacttcaaa ctcgtaccgt gagtaataat gcgccgtcca cggca (SEQ ID NO:37).

The term “anti-miR126” or “anti-miR126 nucleic acid sequence” refers toa sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or100% sequence identity with the perfectly complementary sequence to thetarget miR126 nucleic acid as defined above.

As used herein, the term “miR142” or “miR142 nucleic acid sequence”includes all forms of miR142 including the pri-, pre-, and mature formsof miR142, as well as variants, homologues, modifications, andderivatives thereof (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to the native miR142). Inembodiments, the variants or homologues or derivatives have at least50%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity across the whole sequence or a portion of the sequence(e.g. a 10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, or 80continuous nucleotides portion) compared to a naturally occurring form.In embodiments, the miR142 is the miRNA as identified by NCBI ReferenceSequence: NR 029683.1 or sequence: gacagtgcag tcacccataa agtagaaagcactactaaca gcactggagg gtgtagtgtt tcctacttta tggatgagtg tactgtg (SEQ IDNO: 38).

The term “anti-miR142” or “anti-miR142 nucleic acid sequence” refers toa sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or100% sequence identity with the perfectly complementary sequence to thetarget miR142 nucleic acid as defined above.

As used herein, the term “miR155” or “miR155 nucleic acid sequence”includes all forms of miR155 including the pri-, pre-, and mature formsof miR155, as well as variants, homologues, modifications, andderivatives thereof (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to the native miR155). Inembodiments, the variants or homologues or derivatives have at least50%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity across the whole sequence or a portion of the sequence(e.g. a 10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, or 80continuous nucleotides portion) compared to a naturally occurring form.In embodiments, the miR155 is the miRNA as identified by NCBI ReferenceSequence: NR 030784.1 or sequence: ctgttaatgc taatcgtgat aggggtttttgcctccaact gactcctaca tattagcatt aacag (SEQ ID NO: 39).

The term “anti-miR155” or “anti-miR155 nucleic acid sequence” refers toa sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or100% sequence identity with the perfectly complementary sequence to thetarget miR155 nucleic acid as defined above.

As used herein, the term “miR9” or “miR9 nucleic acid sequence” includesall forms of miR9 including the pri-, pre-, and mature forms of miR9, aswell as variants, homologues, modifications, and derivatives thereof(e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%activity compared to the native miR9). In embodiments, the variants orhomologues or derivatives have at least 50%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity across thewhole sequence or a portion of the sequence (e.g. a 10, 15, 20, 25, 30,40, 45, 50, 55, 60, 65, 70, 75, or 80 continuous nucleotides portion)compared to a naturally occurring form. In embodiments, the miR9 is themiRNA as identified by NCBI Reference Sequence: NR 029691.1, NCBIReference Sequence: NR 029692.1 or sequences: cggggttggt tgttatctttggttatctag ctgtatgagt ggtgtggagt cttcataaag ctagataacc gaaagtaaaaataacccca (SEQ ID NO: 40); ggaggcccgt ttctctcttt ggttatctag ctgtatgagtgccacagagc cgtcataaag ctagataacc gaaagtagaa atgattctca (SEQ ID NO: 41).

The term “anti-miR9” or “anti-miR9 nucleic acid sequence” refers to asequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity with the perfectly complementary sequence to thetarget miR9 nucleic acid as defined above.

As used herein, the term “miR10b” or “miR10b nucleic acid sequence”includes all forms of miR10b including the pri-, pre-, and mature formsof miR10b, as well as variants, homologues, modifications, andderivatives thereof (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to the native miR10b). Inembodiments, the variants or homologues or derivatives have at least50%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity across the whole sequence or a portion of the sequence(e.g. a 10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, or 80continuous nucleotides portion) compared to a naturally occurring form.In embodiments, the miR10b is the miRNA as identified by NCBI ReferenceSequence: NR 029609.1 or sequence: ccagaggttg taacgttgtc tatatataccctgtagaacc gaatttgtgt ggtatccgta tagtcacaga ttcgattcta ggggaatatatggtcgatgc aaaaacttca (SEQ ID NO: 42).

The term “anti-miR10b” or “anti-miR10b nucleic acid sequence” refers toa sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or100% sequence identity with the perfectly complementary sequence to thetarget miR10b nucleic acid as defined above.

As used herein, the term “miR21” “or “miR21 nucleic acid sequence”includes all forms of miR21 including the pri-, pre-, and mature formsof miR21, as well as variants, homologues, modifications, andderivatives thereof (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to the native miR21). In embodiments,the variants or homologues or derivatives have at least 50%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity across the whole sequence or a portion of the sequence (e.g. a10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, or 80 continuousnucleotides portion) compared to a naturally occurring form. Inembodiments, the miR21 is the miRNA as identified by NCBI ReferenceSequence: NR 029493.1 or sequence: tgtcgggtag cttatcagac tgatgttgactgttgaatct catggcaaca ccagtcgatg ggctgtctga ca (SEQ ID NO: 43).

The term “anti-miR21” or “anti-miR21 nucleic acid sequence” refers to asequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity with the perfectly complementary sequence to thetarget miR21 nucleic acid as defined above.

As used herein, the term “miR17” or “miR17 nucleic acid sequence”includes all forms of miR17 including the pri-, pre-, and mature formsof miR17, as well as variants, homologues, modifications, andderivatives thereof (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to the native miR17). In embodiments,the variants or homologues or derivatives have at least 50%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity across the whole sequence or a portion of the sequence (e.g. a10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, or 80 continuousnucleotides portion) compared to a naturally occurring form. Inembodiments, the miR17 is the miRNA as identified by NCBI ReferenceSequence: NR 029487.1 or sequence: gtcagaataa tgtcaaagtg cttacagtgcaggtagtgat atgtgcatct actgcagtga aggcacttgt agcattatgg tgac (SEQ ID NO:44).

The term “anti-miR17” or “anti-miR17 nucleic acid sequence” refers to asequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity with the perfectly complementary sequence to thetarget miR17 nucleic acid as defined above.

As used herein, the term “miR92” or “miR92 nucleic acid sequence”includes all forms of miR92 including the pri-, pre-, and mature formsof miR92, as well as variants, homologues, modifications, andderivatives thereof (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to the native miR92). In embodiments,the variants or homologues or derivatives have at least 50%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity across the whole sequence or a portion of the sequence (e.g. a10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, or 80 continuousnucleotides portion) compared to a naturally occurring form. Inembodiments, the miR92 is the miRNA as identified by NCBI ReferenceSequence: NR 029508.1 or sequence: ctttctacac aggttgggat cggttgcaatgctgtgtttc tgtatggtat tgcacttgtc ccggcctgtt gagtttgg (SEQ ID NO: 45).

The term “anti-miR92” or “anti-miR92 nucleic acid sequence” refers to asequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity with the perfectly complementary sequence to thetarget miR92 nucleic acid as defined above.

As used herein, the term “miR125b” or “miR125b nucleic acid sequence”includes all forms of miR125b including the pri-, pre-, and mature formsof miR125b, as well as variants, homologues, modifications, andderivatives thereof (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to the native miR125b). Inembodiments, the variants or homologues or derivatives have at least50%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity across the whole sequence or a portion of the sequence(e.g. a 10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, or 80continuous nucleotides portion) compared to a naturally occurring form.In embodiments, the miR125b is the miRNA as identified by NCBI ReferenceSequence: NR 029671.1 or sequence: tgcgctcctc tcagtccctg agaccctaacttgtgatgtt taccgtttaa atccacgggt taggctcttg ggagctgcga gtcgtgct (SEQ IDNO: 46).

The term “anti-miR125b” or “anti-miR125b nucleic acid sequence” refersto a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or100% sequence identity with the perfectly complementary sequence to thetarget miR125b nucleic acid as defined above.

As used herein, the term “miR146a” or “miR146 nucleic acid sequence”includes all forms of miR146a including the pri-, pre-, and mature formsof miR146a, as well as variants, homologues, modifications, andderivatives thereof (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to the native miR146a). Inembodiments, the variants or homologues or derivatives have at least50%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity across the whole sequence or a portion of the sequence(e.g. a 10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, or 80continuous nucleotides portion) compared to a naturally occurring form.In embodiments, the miR146a is the miRNA as identified by NCBI ReferenceSequence: NR 029701.1 or sequence: ccgatgtgta tcctcagctt tgagaactgaattccatggg ttgtgtcagt gtcagacctc tgaaattcag ttcttcagct gggatatctctgtcatcgt (SEQ ID NO: 47).

The term “anti-miR146a” or “anti-miR146a nucleic acid sequence” refersto a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or100% sequence identity with the perfectly complementary sequence to thetarget miR146a nucleic acid as defined above.

As used herein, the term “microRNA-mimic (miRNA-mimic)” or “miRNA-mimicnucleic acid sequence” is used according to its plain and ordinarymeaning and refers to single, double or triple stranded oligonucleotidethat is capable of effecting a biological function similar to amicroRNA. In embodiments, miRNA-mimic may be non-natural double-strandedmiR-like RNA fragments. Such an RNA fragment may be designed to have its5′-end bearing a partially complementary motif to the selected sequencein the 3′UTR unique to the target gene. Once introduced into cells, thisRNA fragment, may mimic an endogenous miRNA, bind specifically to itstarget gene and produce posttranscriptional repression, morespecifically translational inhibition, of the gene. Unlike endogenousmiRNAs, miRNA-mimics may act in a gene-specific fashion. In embodiments,the miRNA-mimic may be a double stranded oligomer of 20-30 bases (e.g.,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 bases). In embodiments, themiRNA-mimic may be a triple stranded oligomer of 20-30 bases (e.g., 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30 bases). In embodiments, themiRNA-mimic may have a 2′chemical modification. In embodiments, themiRNA-mimic may have serum stability-enhancing chemical modification,e.g., a phosphothioate internucleotide linkage, a 2′-O-methylribonucleotide, a 2′-deoxy-2′fluoro ribonucleotide, a 2′-deoxyribonucleotide, a universal base nucleotide, a 5-C methyl nucleotide, aninverted deoxybasic residue incorporation, or a locked nucleic acid.

As used herein, the term “miR126-mimic” or “miR26-mimic nucleic acidsequence” refers to an oligonucleotide that is structurallysubstantially similar to miR126 and is capable of effecting a biologicalfunction similar to miR126. In embodiments, the miR126-mimic has atleast 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity comparedto the native miR126. In embodiments, the miR126-mimic has at least 50%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity across the whole sequence or a portion of the sequence(e.g. a 10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, or 80continuous nucleotides portion) compared to native miR126.

As used herein, the term “miR142-mimic” or “miR142-mimic nucleic acidsequence” refers to an oligonucleotide that is structurallysubstantially similar to miR142 and is capable of effecting a biologicalfunction similar to miR142. In embodiments, the miR142-mimic has atleast 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity comparedto the native miR142. In embodiments, the miR142-mimic has at least 50%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity across the whole sequence or a portion of the sequence(e.g. a 10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, or 80continuous nucleotides portion) compared to native miR142.

As used herein, the term “miR155-mimic” or “miR155-mimic nucleic acidsequence” refers to an oligonucleotide that is structurallysubstantially similar to miR155 and is capable of effecting a biologicalfunction similar to miR155. In embodiments, the miR155-mimic has atleast 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity comparedto the native miR155. In embodiments, the miR155-mimic has at least 50%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity across the whole sequence or a portion of the sequence(e.g. a 10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, or 80continuous nucleotides portion) compared to native miR155.

As used herein, the term “miR9-mimic” or “miR9-mimic nucleic acidsequence” refers to an oligonucleotide that is structurallysubstantially similar to miR9 and is capable of effecting a biologicalfunction similar to miR9. In embodiments, the miR9-mimic has at least50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to thenative miR9. In embodiments, the miR9-mimic has at least 50%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity across the whole sequence or a portion of the sequence (e.g. a10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, or 80 continuousnucleotides portion) compared to native miR9.

As used herein, the term “miR10b-mimic” or “miR10b-mimic nucleic acidsequence” refers to an oligonucleotide that is structurallysubstantially similar to miR10b and is capable of effecting a biologicalfunction similar to miR10b. In embodiments, the miR10b-mimic has atleast 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity comparedto the native miR10b. In embodiments, the miR10b-mimic has at least 50%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity across the whole sequence or a portion of the sequence(e.g. a 10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, or 80continuous nucleotides portion) compared to native miR10b.

As used herein, the term “miR21-mimic” or “miR21-mimic nucleic acidsequence” refers to an oligonucleotide that is structurallysubstantially similar to miR21 and is capable of effecting a biologicalfunction similar to miR21. In embodiments, the miR21-mimic has at least50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to thenative miR21. In embodiments, the miR21-mimic has at least 50%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity across the whole sequence or a portion of the sequence (e.g. a10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, or 80 continuousnucleotides portion) compared to native miR21.

As used herein, the term “miR17-mimic” or “miR17-mimic nucleic acidsequence” refers to an oligonucleotide that is structurallysubstantially similar to miR17 and is capable of effecting a biologicalfunction similar to miR17. In embodiments, the miR17-mimic has at least50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to thenative miR17. In embodiments, the miR17-mimic has at least 50%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity across the whole sequence or a portion of the sequence (e.g. a10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, or 80 continuousnucleotides portion) compared to native miR17.

As used herein, the term “miR92-mimic” or “miR92-mimic nucleic acidsequence” refers to an oligonucleotide that is structurallysubstantially similar to miR92 and is capable of effecting a biologicalfunction similar to miR92. In embodiments, the miR92-mimic has at least50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to thenative miR92. In embodiments, the miR92-mimic has at least 50%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity across the whole sequence or a portion of the sequence (e.g. a10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, or 80 continuousnucleotides portion) compared to native miR92.

As used herein, the term “miR125b-mimic” or “miR125b-mimic nucleic acidsequence” refers to an oligonucleotide that is structurallysubstantially similar to miR125b and is capable of effecting abiological function similar to miR125b. In embodiments, themiR125b-mimic has at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or100% activity compared to the native miR125b. In embodiments, themiR125b-mimic has at least 50%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% sequence identity across the whole sequenceor a portion of the sequence (e.g. a 10, 15, 20, 25, 30, 40, 45, 50, 55,60, 65, 70, 75, or 80 continuous nucleotides portion) compared to nativemiR125b.

As used herein, the term “miR146a-mimic” or “miR146a-mimic nucleic acidsequence” refers to an oligonucleotide that is structurallysubstantially similar to miR146a and is capable of effecting abiological function similar to miR146a. In embodiments, themiR146a-mimic has at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or100% activity compared to the native miR146a. In embodiments, themiR146a-mimic has at least 50%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% sequence identity across the whole sequenceor a portion of the sequence (e.g. a 10, 15, 20, 25, 30, 40, 45, 50, 55,60, 65, 70, 75, or 80 continuous nucleotides portion) compared to nativemiR146a.

As used herein, the term “phosphorothioated oligodeoxynucleotide (ODN)”refers to a nucleic acid sequence, e.g., “CpG nucleic acid sequence” or“GpC nucleic acid sequence”, in which some or all the internucleotidelinkages constitute a phosphorothioate linkage. In embodiments,phosphorothioated oligodeoxynucleotide (ODN) is 15 to 30 bases long,single-stranded, partly or completely phosphorothioated. The partlyphosphorothioated ODN is an ODN in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or28, internucleotide linkages constitute a phosphorothioate linkage.

In embodiments, the term “CpG motif” in a nucleic acid refers to anucleic acid in which a 5′ C nucleotide connected to a 3′ G nucleotidethrough a phosphodiester internucleotide linkage or a phosphodiesterderivative internucleotide linkage. In embodiments, the term “CpG motif”in a nucleic acid refers to a nucleic acid in which a 5′ G nucleotideconnected to a 3′ C nucleotide through a phosphodiester internucleotidelinkage or a phosphodiester derivative internucleotide linkage (aka a“GpC nucleic acid sequence). In embodiments, a CpG motif includes aphosphodiester internucleotide linkage. In embodiments, a CpG motifincludes a phosphodiester derivative internucleotide linkage. Inembodiments, a CpG motif includes a phosphorothioate linkage.

As used herein, the term “Class A CpG ODN” or “A-class CpG ODN” or“D-type CpG ODN” or “Class A CpG DNA sequence” is used in accordancewith its common meaning in the biological and chemical sciences andrefers to a CpG motif including oligodeoxynucleotide including one ormore of poly-G sequence at the 5′, 3′, or both ends; an internalpalindrome sequence including CpG motif; or one or more phosphodiesterderivatives linking deoxynucleotides. In embodiments, a Class A CpG ODNincludes poly-G sequence at the 5′, 3′, or both ends; an internalpalindrome sequence including CpG motif; and one or more phosphodiesterderivatives linking deoxynucleotides. In embodiments, the phosphodiesterderivative is phosphorothioate. Examples of Class A CpG ODNs include ODND19, ODN 1585, ODN 2216, and ODN 2336.

As used herein, the term “Class B CpG ODN” or “B-class CpG ODN” or“K-type CpG ODN” or “Class B CpG DNA sequence” is used in accordancewith its common meaning in the biological and chemical sciences andrefers to a CpG motif including oligodeoxynucleotide including one ormore of a 6mer motif including a CpG motif; phosphodiester derivativeslinking all deoxynucleotides. In embodiments, a 6mer motif comprises5′-PuPyCGPyPu-3′ (SEQ ID NO: 15), where Pu represents a purinecontaining nucleobase (e.g., A or G) and Py represents a pyrimidinecontaining nucleobase (e.g., T/U or C). In embodiments, a Class B CpGODN includes one or more copies of a 6mer motif including a CpG motifand phosphodiester derivatives linking all deoxynucleotides. Inembodiments, the phosphodiester derivative is phosphorothioate. Inembodiments, a Class B CpG ODN includes one 6mer motif including a CpGmotif. In embodiments, a Class B CpG ODN includes two copies of a 6mermotif including a CpG motif. In embodiments, a Class B CpG ODN includesthree copies of a 6mer motif including a CpG motif. In embodiments, aClass B CpG ODN includes four copies of a 6mer motif including a CpGmotif. Examples of Class B CpG ODNs include ODN 1668, ODN 1826, ODN2006, and ODN 2007.

As used herein, the term “Class C CpG ODN” or “C-class CpG ODN” “or“C-type CpG DNA sequence” is used in accordance with its common meaningin the biological and chemical sciences and refers to anoligodeoxynucleotide including a palindrome sequence including a CpGmotif and phosphodiester derivatives (phosphorothioate) linking alldeoxynucleotides. Examples of Class C CpG ODNs include ODN 2395 and ODNM362.

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchednon-cyclic carbon chain (or carbon), or combination thereof, which maybe fully saturated, mono- or polyunsaturated and can include di- andmultivalent radicals, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, (cyclohexyl)methyl, homologs and isomers of, for example,n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkylgroup is one having one or more double bonds or triple bonds. Examplesof unsaturated alkyl groups include, but are not limited to, vinyl,2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and thehigher homologs and isomers. An alkoxy is an alkyl attached to theremainder of the molecule via an oxygen linker (—O—).

The term “alkylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkyl, asexemplified, but not limited by, —CH₂CH₂CH₂—. Typically, an alkyl (oralkylene) group will have from 1 to 24 carbon atoms. A “lower alkyl” or“lower alkylene” is a shorter chain alkyl or alkylene group, generallyhaving eight or fewer carbon atoms. The term “alkenylene,” by itself oras part of another substituent, means, unless otherwise stated, adivalent radical derived from an alkene.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable non-cyclic straight or branchedchain, or combinations thereof, including at least one carbon atom andat least one heteroatom selected from the group consisting of O, N, P,Si, and S, and wherein the nitrogen and sulfur atoms may optionally beoxidized, and the nitrogen heteroatom may optionally be quaternized. Theheteroatom(s) 0, N, P, S, and Si may be placed at any interior positionof the heteroalkyl group or at the position at which the alkyl group isattached to the remainder of the molecule. Examples include, but are notlimited to: —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃,—Si(CH₃)₃, —CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, —O—CH₃, —O—CH₂—CH₃, and—CN. Up to two or three heteroatoms may be consecutive, such as, forexample, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like Still further, for alkylene and heteroalkylene linking groups,no orientation of the linking group is implied by the direction in whichthe formula of the linking group is written. For example, the formula—C(O)₂R′— represents both —C(O)₂R′- and —R′C(O)₂—. As described above,heteroalkyl groups, as used herein, include those groups that areattached to the remainder of the molecule through a heteroatom, such as—C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SR′, and/or —SO₂R′. Where“heteroalkyl” is recited, followed by recitations of specificheteroalkyl groups, such as —NR′R″ or the like, it will be understoodthat the terms heteroalkyl and —NR′R″ are not redundant or mutuallyexclusive. Rather, the specific heteroalkyl groups are recited to addclarity. Thus, the term “heteroalkyl” should not be interpreted hereinas excluding specific heteroalkyl groups, such as —NR′R″ or the like.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, mean, unless otherwise stated, cyclicnon-aromatic versions of “alkyl” and “heteroalkyl,” respectively,wherein the carbons making up the ring or rings do not necessarily needto be bonded to a hydrogen due to all carbon valencies participating inbonds with non-hydrogen atoms. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a“heterocycloalkylene,” alone or as part of another substituent, means adivalent radical derived from a cycloalkyl and heterocycloalkyl,respectively.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” includes, but is not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “acyl” means, unless otherwise stated, —C(O)R where R is asubstituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings) that are fused together(i.e., a fused ring aryl) or linked covalently (e.g., biphenyl). A fusedring aryl refers to multiple rings fused together wherein at least oneof the fused rings is an aryl ring. The term “heteroaryl” refers to arylgroups (or rings) that contain at least one heteroatom such as N, O, orS, wherein the nitrogen and sulfur atoms are optionally oxidized, andthe nitrogen atom(s) are optionally quaternized. Thus, the term“heteroaryl” includes fused ring heteroaryl groups (i.e., multiple ringsfused together wherein at least one of the fused rings is aheteroaromatic ring). A 5,6-fused ring heteroarylene refers to two ringsfused together, wherein one ring has 5 members and the other ring has 6members, and wherein at least one ring is a heteroaryl ring. Likewise, a6,6-fused ring heteroarylene refers to two rings fused together, whereinone ring has 6 members and the other ring has 6 members, and wherein atleast one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylenerefers to two rings fused together, wherein one ring has 6 members andthe other ring has 5 members, and wherein at least one ring is aheteroaryl ring. A heteroaryl group can be attached to the remainder ofthe molecule through a carbon or heteroatom. Non-limiting examples ofaryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below. An “arylene” and a“heteroarylene,” alone or as part of another substituent, mean adivalent radical derived from an aryl and heteroaryl, respectively.Non-limiting examples of heteroaryl groups include pyridinyl,pyrimidinyl, thiophenyl, thienyl, furanyl, indolyl, benzoxadiazolyl,benzodioxolyl, benzodioxanyl, thianaphthanyl, pyrrolopyridinyl,indazolyl, quinolinyl, quinoxalinyl, pyridopyrazinyl, quinazolinonyl,benzoisoxazolyl, imidazopyridinyl, benzofuranyl, benzothienyl,benzothiophenyl, phenyl, naphthyl, biphenyl, pyrrolyl, pyrazolyl,imidazolyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl, furylthienyl,pyridyl, pyrimidyl, benzothiazolyl, purinyl, benzimidazolyl,isoquinolyl, thiadiazolyl, oxadiazolyl, pyrrolyl, diazolyl, triazolyl,tetrazolyl, benzothiadiazolyl, isothiazolyl, pyrazolopyrimidinyl,pyrrolopyrimidinyl, benzotriazolyl, benzoxazolyl, or quinolyl. Theexamples above may be substituted or unsubstituted and divalent radicalsof each heteroaryl example above are non-limiting examples ofheteroarylene.

A fused ring heterocyloalkyl-aryl is an aryl fused to aheterocycloalkyl. A fused ring heterocycloalkyl-heteroaryl is aheteroaryl fused to a heterocycloalkyl. A fused ringheterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl.A fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkylfused to another heterocycloalkyl. Fused ring heterocycloalkyl-aryl,fused ring heterocycloalkyl-heteroaryl, fused ringheterocycloalkyl-cycloalkyl, or fused ringheterocycloalkyl-heterocycloalkyl may each independently beunsubstituted or substituted with one or more of the substitutentsdescribed herein.

The term “oxo,” as used herein, means an oxygen that is double bonded toa carbon atom.

The term “alkylsulfonyl,” as used herein, means a moiety having theformula —S(O₂)—R′, where R′ is a substituted or unsubstituted alkylgroup as defined above. R′ may have a specified number of carbons (e.g.,“C₁-C₄ alkylsulfonyl”).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl,” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″,—ONR′R″, —NR′C═(O)NR″NR′″R″″, —CN, —NO₂, in a number ranging from zeroto (2m′+1), where m′ is the total number of carbon atoms in suchradical. R, R′, R″, R′″, and R″″ each preferably independently refer tohydrogen, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl (e.g., aryl substituted with 1-3halogens), substituted or unsubstituted heteroaryl, substituted orunsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″, and R″″ group when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 4-, 5-, 6-, or 7-memberedring. For example, —NR′R″ includes, but is not limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″,—OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″, —ONR′R″,—NR′C═(O)NR″NR′″R″″, —CN, NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy,and fluoro(C₁-C₄)alkyl, in a number ranging from zero to the totalnumber of open valences on the aromatic ring system; and where R′, R″,R′″, and R″″ are preferably independently selected from hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″, and R″″ groupswhen more than one of these groups is present.

Two or more substituents may optionally be joined to form aryl,heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-calledring-forming substituents are typically, though not necessarily, foundattached to a cyclic base structure. In one embodiment, the ring-formingsubstituents are attached to adjacent members of the base structure. Forexample, two ring-forming substituents attached to adjacent members of acyclic base structure create a fused ring structure. In anotherembodiment, the ring-forming substituents are attached to a singlemember of the base structure. For example, two ring-forming substituentsattached to a single member of a cyclic base structure create aspirocyclic structure. In yet another embodiment, the ring-formingsubstituents are attached to non-adjacent members of the base structure.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally form a ring of the formula -T-C(O)—(CRR′)q-U—, wherein Tand U are independently —NR—, —O—, —CRR′—, or a single bond, and q is aninteger of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)r-B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′—, or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′— (C″R″R′″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″, and R′″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl.

As used herein, the terms “heteroatom” or “ring heteroatom” are meant toinclude, oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

-   -   (A) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,        —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,        —NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH,        —NHOH, —OCF₃, —OCHF₂, —NHSO₂CH₃, —N₃, unsubstituted alkyl,        unsubstituted heteroalkyl, unsubstituted cycloalkyl,        unsubstituted heterocycloalkyl, unsubstituted aryl,        unsubstituted heteroaryl, and    -   (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,        heteroaryl, substituted with at least one substituent selected        from:

(i) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH,—SO₂C₁, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, —NHC═(O)NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂, —NHSO₂CH₃,—N₃, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl,unsubstituted heteroaryl, and

-   -   (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,        heteroaryl, substituted with at least one substituent selected        from:    -   (a) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,        —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,        —NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH,        —NHOH, —OCF₃, —OCHF₂, —NHSO₂CH₃, —N₃, unsubstituted alkyl,        unsubstituted heteroalkyl, unsubstituted cycloalkyl,        unsubstituted heterocycloalkyl, unsubstituted aryl,        unsubstituted heteroaryl, and    -   (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,        heteroaryl, substituted with at least one substituent selected        from: oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,        —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,        —NHC—(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH,        —NHOH, —OCF₃, —OCHF₂, —NHSO₂CH₃, —N₃, unsubstituted alkyl,        unsubstituted heteroalkyl, unsubstituted cycloalkyl,        unsubstituted heterocycloalkyl, unsubstituted aryl,        unsubstituted heteroaryl.

A “size-limited substituent” or “size-limited substituent group,” asused herein, means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl.

A “lower substituent” or “lower substituent group,” as used herein,means a group selected from all of the substituents described above fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl.

In some embodiments, each substituted group described in the compoundsherein is substituted with at least one substituent group. Morespecifically, in some embodiments, each substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, substituted heteroaryl, substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene described in the compounds herein are substituted with atleast one substituent group. In other embodiments, at least one or allof these groups are substituted with at least one size-limitedsubstituent group. In other embodiments, at least one or all of thesegroups are substituted with at least one lower substituent group.

In other embodiments of the compounds herein, each substituted orunsubstituted alkyl may be a substituted or unsubstituted C₁-C₂₀ alkyl,each substituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and/or each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl. In someembodiments of the compounds herein, each substituted or unsubstitutedalkylene is a substituted or unsubstituted C₁-C₂₀ alkylene, eachsubstituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 20 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₈cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 8 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedC₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroaryleneis a substituted or unsubstituted 5 to 10 membered heteroarylene.

In some embodiments, each substituted or unsubstituted alkyl is asubstituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and/or each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl. In some embodiments, each substituted orunsubstituted alkylene is a substituted or unsubstituted C₁-C₈ alkylene,each substituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 8 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₇cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 7 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedC₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroaryleneis a substituted or unsubstituted 5 to 9 membered heteroarylene. In someembodiments, the compound is a chemical species set forth in theExamples section below.

As used herein, the term “conjugated” when referring to two moietiesmeans the two moieties are bonded, wherein the bond or bonds connectingthe two moieties may be covalent or non-covalent. In embodiments, thetwo moieties are covalently bonded to each other (e.g., directly orthrough a covalently bonded intermediary). In embodiments, the twomoieties are non-covalently bonded (e.g., through ionic bond(s), van derwaal's bond(s)/interactions, hydrogen bond(s), polar bond(s), orcombinations or mixtures thereof).

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical,magnetic resonance imaging, or other physical means. For example, usefuldetectable moieties include 32P, fluorescent dyes, electron-densereagents, enzymes (e.g., as commonly used in an ELISA), biotin,digoxigenin, paramagnetic molecules, paramagnetic nanoparticles,ultrasmall superparamagnetic iron oxide (“USPIO”) nanoparticles, USPIOnanoparticle aggregates, superparamagnetic iron oxide (“SPIO”)nanoparticles, SPIO nanoparticle aggregates, monochrystalline SPIO,monochrystalline SPIO aggregates, monochrystalline iron oxidenanoparticles, monochrystalline iron oxide, other nanoparticle contrastagents, liposomes or other delivery vehicles containing Gadoliniumchelate (“Gd-chelate”) molecules, Gadolinium, radioisotopes,radionuclides (e.g., carbon-11, nitrogen-13, oxygen-15, fluorine-18,rubidium-82), fluorodeoxyglucose (e.g., fluorine-18 labeled), any gammaray emitting radionuclides, positron-emitting radionuclide, radiolabeledglucose, radiolabeled water, radiolabeled ammonia, biocolloids,microbubbles (e.g., including microbubble shells including albumin,galactose, lipid, and/or polymers; microbubble gas core including air,heavy gas(es), perfluorcarbon, nitrogen, octafluoropropane, perflexanelipid microsphere, perflutren, etc.), iodinated contrast agents (e.g.,iohexol, iodixanol, ioversol, iopamidol, ioxilan, iopromide,diatrizoate, metrizoate, ioxaglate), barium sulfate, thorium dioxide,gold, gold nanoparticles, gold nanoparticle aggregates, fluorophores,two-photon fluorophores, or haptens and proteins or other entities whichcan be made detectable, e.g., by incorporating a radiolabel into apeptide or antibody specifically reactive with a target peptide.Detectable moieties also include any of the above compositionsencapsulated in nanoparticles, particles, aggregates, coated withadditional compositions, derivatized for binding to a targeting agent(e.g., compound described herein). Any method known in the art forconjugating an oligonucleotide or protein to the label may be employed,e.g., using methods described in Hermanson, Bioconjugate Techniques1996, Academic Press, Inc., San Diego.

A “cell” as used herein, refers to a cell carrying out metabolic orother function sufficient to preserve or replicate its genomic DNA. Acell can be identified by well-known methods in the art including, forexample, presence of an intact membrane, staining by a particular dye,ability to produce progeny or, in the case of a gamete, ability tocombine with a second gamete to produce a viable offspring. Cells mayinclude prokaryotic and eukaryotic cells. Prokaryotic cells include butare not limited to bacteria. Eukaryotic cells include but are notlimited to yeast cells and cells derived from plants and animals, forexample mammalian, insect (e.g., spodoptera) and human cells. The term“cell” as used herein also refers to individual cells, cell lines, orcultures derived from such cells. A “culture” refers to a compositioncomprising isolated cells of the same or a different type.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or higher identity over a specified region whencompared and aligned for maximum correspondence over a comparison windowor designated region) as measured using a BLAST or BLAST 2.0 sequencecomparison algorithms with default parameters described below, or bymanual alignment and visual inspection (see, e.g., NCBI web site or thelike). Such sequences are then said to be “substantially identical.”This definition also refers to, or may be applied to, the compliment ofa test sequence. The definition also includes sequences that havedeletions and/or additions, as well as those that have substitutions. Asdescribed below, the preferred algorithms can account for gaps and thelike. Preferably, identity exists over a region that is at least about10 amino acids or 20 nucleotides in length, or more preferably over aregion that is 10-50 amino acids or 20-50 nucleotides in length. As usedherein, percent (%) nucleic acid sequence identity is defined as thepercentage of nucleotides in a candidate sequence that are identical tothe nucleotides in a reference sequence, after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percentsequence identity. Alignment for purposes of determining percentsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR)software. Appropriate parameters for measuring alignment, including anyalgorithms needed to achieve maximal alignment over the full-length ofthe sequences being compared can be determined by known methods.

For sequence comparisons, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative t″o thereference sequence, based on the program parameters.

The term “complementary” or “complementarity” refers to the ability of anucleic acid to form hydrogen bond(s) with another nucleic acid sequenceby either traditional Watson-Crick or other non-traditional types. Forexample, the sequence A-G-T is complementary to the sequence T-C-A. Apercent complementarity indicates the percentage of residues in anucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crickbase pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9,10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).“Perfectly complementary” means that all the contiguous residues of anucleic acid sequence will hydrogen bond with the same number ofcontiguous residues in a second nucleic acid sequence. “Substantiallycomplementary” as used herein refers to a degree of complementarity thatis at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. 97%, 98%, 99%, or100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refersto two nucleic acids that hybridize under stringent conditions.

As used herein, “stringent conditions” for hybridization refer toconditions under which a nucleic acid having complementarity to a targetsequence predominantly hybridizes with the target sequence, andsubstantially does not hybridize to non-target sequences. Stringentconditions are generally sequence-dependent, and vary depending on anumber of factors. In general, the longer the sequence, the higher thetemperature at which the sequence specifically hybridizes to its targetsequence. Non-limiting examples of stringent conditions are described indetail in Tijssen (1993), Laboratory Techniques In Biochemistry AndMolecular Biology-Hybridization With Nucleic Acid Probes Part 1, SecondChapter “Overview of principles of hybridization and the strategy ofnucleic acid probe assay”, Elsevier, N.Y.

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson Crick base pairing, Hoogstein binding, or inany other sequence specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming a multistranded complex, a single self 17 hybridizing strand, or anycombination of these. A hybridization reaction may constitute a step ina more extensive process, such as the initiation of PCR, or the cleavageof a polynucleotide by an enzyme. A sequence capable of hybridizing witha given sequence is referred to as the “complement” of the givensequence.

“Patient,” “subject,” “patient in need thereof,” and “subject in needthereof” are herein used interchangeably and refer to a living organismsuffering from or prone to a disease or condition that can be treated byadministration using the methods and compositions provided herein.Non-limiting examples include humans, other mammals, bovines, rats,mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammaliananimals. In some embodiments, a patient is human. Tissues, cells andtheir progeny of a biological entity obtained in vitro or cultured invitro are also contemplated.

As used herein, the term “administering” means oral administration,administration as a suppository, topical contact, intravenous,parenteral, intraperitoneal, intramuscular, intralesional, intrathecal,intranasal or subcutaneous administration, or the implantation of aslow-release device, e.g., a mini-osmotic pump, to a subject.Administration is by any route, including parenteral and transmucosal(e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, ortransdermal). Parenteral administration includes, e.g., intravenous,intramuscular, intra-arteriole, intradermal, subcutaneous,intraperitoneal, intraventricular, and intracranial. Other modes ofdelivery include, but are not limited to, the use of liposomalformulations, intravenous infusion, transdermal patches, etc.

The terms “treat,” “treating” or “treatment,” and other grammaticalequivalents as used herein, include alleviating, abating, ameliorating,or preventing a disease, condition or symptoms, preventing additionalsymptoms, ameliorating or preventing the underlying metabolic causes ofsymptoms, inhibiting the disease or condition, e.g., arresting thedevelopment of the disease or condition, relieving the disease orcondition, causing regression of the disease or condition, relieving acondition caused by the disease or condition, or stopping the symptomsof the disease or condition, and are intended to include prophylaxis.The terms further include achieving a therapeutic benefit and/or aprophylactic benefit. By therapeutic benefit is meant eradication oramelioration of the underlying disorder being treated. Also, atherapeutic benefit is achieved with the eradication or amelioration ofone or more of the physiological symptoms associated with the underlyingdisorder such that an improvement is observed in the patient,notwithstanding that the patient may still be afflicted with theunderlying disorder.

The terms “prevent,” “preventing,” or “prevention,” and othergrammatical equivalents as used herein, include to keep from developing,occur, hinder or avert a disease or condition symptoms as well as todecrease the occurrence of symptoms. The prevention may be complete(i.e., no detectable symptoms) or partial, so that fewer symptoms areobserved than would likely occur absent treatment. The terms furtherinclude a prophylactic benefit. For a disease or condition to beprevented, the compositions may be administered to a patient at risk ofdeveloping a particular disease, or to a patient reporting one or moreof the physiological symptoms of a disease, even though a diagnosis ofthis disease may not have been made.

The term “inhibiting” also means reducing an effect (disease state orexpression level of a gene/protein/mRNA) relative to the state in theabsence of a compound or composition of the present disclosure.

A “test compound” as used herein refers to an experimental compound usedin a screening process to identify activity, non-activity, or othermodulation of a particularized biological target or pathway.

“Control” or “control experiment” is used in accordance with its plainordinary meaning and refers to an experiment in which the subjects orreagents of the experiment are treated as in a parallel experimentexcept for omission of a procedure, reagent, or variable of theexperiment. In some instances, the control is used as a standard ofcomparison in evaluating experimental effects. In some embodiments, acontrol is the measurement of the activity of a protein in the absenceof a compound as described herein (including embodiments and examples).

“Disease” or “condition” refer to a state of being or health status of apatient or subject capable of being treated with the compounds ormethods provided herein. In some instances, “disease” or “condition”refers to a “cancer”.

As used herein, the term “cancer” refers to all types of cancer,neoplasm, malignant or benign tumors found in mammals, includingleukemia, carcinomas and sarcomas. Exemplary cancers include breastcancer, ovarian cancer, colon cancer, liver cancer, kidney cancer andpancreatic cancer. Additional examples include leukemia (e.g., acutemyeloid leukemia (“AML”) or chronic myeloid leukemia (“CML”)), cancer ofthe brain, lung cancer, non-small cell lung cancer, melanoma, sarcomas,and prostate cancer, cervix cancers, stomach cancers, head & neckcancers, uterus cancers, mesothelioma, metastatic bone cancer,Medulloblastoma, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiplemyeloma, neuroblastoma, rhabdomyosarcoma, primary thrombocytosis,primary macroglobulinemia, primary brain tumors, malignant pancreaticinsulanoma, malignant carcinoid, urinary bladder cancer, premalignantskin lesions, testicular cancer, lymphomas, thyroid cancer,neuroblastoma, esophageal cancer, genitourinary tract cancer, malignanthypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms ofthe endocrine and exocrine pancreas.

An autoimmune disease refers to a disease in which the body's immunesystem attacks healthy cells. Examples of autoimmune diseases include,but are not limited to, rheumatoid arthritis, psoriasis, systemic lupuserythematosus (SLE), type II diabetes, non-alcoholic fatty liverdisease, non-alcoholic steatohepatitis, multiple sclerosis (MS),Parkinson's disease (PD, Alzheimer's disease (AD) and inflammatory boweldisease (IBD).

An infectious disease refers to a medical condition caused by the growthand spread of harmful organisms (e.g., bacteria, viruses, fungi orparasites) within the body. Examples of infectious diseases include, butare not limited to, tuberculosis, influenza, Ebola, HIV, HPV infectionand hepatitis.

“Contacting” is used in accordance with its plain ordinary meaning andrefers to the process of allowing at least two distinct species (e.g.,chemical compounds including biomolecules or cells) to becomesufficiently proximal to react, interact or physically touch. It shouldbe appreciated; however, the resulting reaction product can be produceddirectly from a reaction between the added reagents or from anintermediate from one or more of the added reagents which can beproduced in the reaction mixture. In some embodiments contactingincludes allowing a compound described herein to interact with a proteinor enzyme.

The terms “phenotype” and “phenotypic” as used herein refer to anorganism's observable characteristics such as onset or progression ofdisease symptoms, biochemical properties, or physiological properties.

The word “expression” or “expressed” as used herein in reference to aDNA nucleic acid sequence (e.g., a gene) means the transcriptionaland/or translational product of that sequence. The level of expressionof a DNA molecule in a cell may be determined on the basis of either theamount of corresponding mRNA that is present within the cell or theamount of protein encoded by that DNA produced by the cell (Sambrook etal., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88). When usedin reference to polypeptides, expression includes any step involved inthe production of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion. Expression can bedetected using conventional techniques for detecting protein (e.g.,ELISA, Western blotting, flow cytometry, immunofluorescence,immunohistochemistry, etc.).

The term “gene” means the segment of DNA involved in producing aprotein; it includes regions preceding and following the coding region(leader and trailer) as well as intervening sequences (introns) betweenindividual coding segments (exons). The leader, the trailer as well asthe introns include regulatory elements that are necessary during thetranscription and the translation of a gene. Further, a “protein geneproduct” is a protein expressed from a particular gene.

For specific proteins described herein (e.g., CD34 or CD38), the namedprotein includes any of the protein's naturally occurring forms,variants or homologs (e.g., within at least 50%, 80%, 90%, 95%, 96%,97%, 98%, 99% or 100% activity compared to the native protein). In someembodiments, variants or homologs have at least 90%, 95%, 96%, 97%, 98%,99% or 100% amino acid sequence identity across the whole sequence or aportion of the sequence (e.g. a 50, 100, 150 or 200 continuous aminoacid portion) compared to a naturally occurring form. In otherembodiments, the protein is the protein as identified by its NCBIsequence reference. In other embodiments, the protein is the protein asidentified by its NCBI sequence reference, homolog or functionalfragment thereof.

The term “CD34” refers to hematopoietic progenitor cell antigen CD34also known as CD34 antigen that is encoded by the CD34 gene in humans.It is a cell surface glycoprotein and functions as a cell-cell adhesionfactor. The term “CD34” as provided herein includes any of the CD34protein naturally occurring forms, homologs or variants that maintainthe activity of CD34 (e.g., within at least 50%, 80%, 90%, 95%, 96%,97%, 98%, 99% or 100% activity compared to the native protein). Inembodiments, variants or homologs have at least 50%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequenceidentity across the whole sequence or a portion of the sequence (e.g. a50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring form. In embodiments, the CD34 protein is theprotein as identified by the NCBI sequence reference NP 001764 or NP001020280.1, homolog or functional fragment thereof.

The term “CD38” refers to cluster of differentiation 38, also known ascyclic ADP ribose hydrolase that is encoded by the CD38 gene in humans.It is a cell surface glycoprotein and functions in cell-cell adhesionand signaling transduction. The term “CD38” as provided herein includesany of the CD38 protein naturally occurring forms, homologs or variantsthat maintain the activity of CD38 (e.g., within at least 50%, 80%, 90%,95%, 96%, 97%, 98%, 99% or 100% activity compared to the nativeprotein). In embodiments, variants or homologs have at least 50%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity across the whole sequence or a portion of the sequence(e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring form. In embodiments, the CD38 protein is theprotein as identified by the NCBI sequence reference NP 001766.2,homolog or functional fragment thereof

The term “an amount of” in reference to a polynucleotide or polypeptide,refers to an amount at which a component or element is detected. Theamount may be measured against a control, for example, wherein anincreased level of a particular polynucleotide or polypeptide inrelation to the control, demonstrates enrichment of the polynucleotideor polypeptide. Thus, in embodiments, an increased amount indicates agreater level or efficiency of grafting HSPCs described herein into ahost (e.g., mouse). The term refers to quantitative measurement of theenrichment as well as qualitative measurement of an increase or decreaserelative to a control.

Throughout the description and claims of this specification the word“comprise” and other forms of the word, such as “comprising” and“comprises,” means including but not limited to, and is not intended toexclude, for example, other components.

“Analog,” “analogue,” or “derivative” is used in accordance with itsplain ordinary meaning within Chemistry and Biology and refers to achemical agent that is structurally similar to another agent (i.e., aso-called “reference” agent) but differs in composition, e.g., in thereplacement of one atom by an atom of a different element, or in thepresence of a particular functional group, or the replacement of onefunctional group by another functional group, or the absolutestereochemistry of a chiral center of the reference agent. In someembodiments, a derivative may be a conjugate with a pharmaceuticallyacceptable agent, for example, phosphate or phosphonate.

As used herein, the term “salt” refers to acid or base salts of theagents used herein. Illustrative but non-limiting examples of acceptablesalts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoricacid, sulfuric acid, and the like) salts, organic acid (acetic acid,propionic acid, glutamic acid, citric acid, and the like) salts, andquaternary ammonium (methyl iodide, ethyl iodide, and the like) salts.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds that are prepared with relatively nontoxic acidsor bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present disclosurecontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentdisclosure contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, e.g., Berge et al., Journal of Pharmaceutical Science 66:1-19(1977)). Certain specific compounds of the present disclosure containboth basic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts. Otherpharmaceutically acceptable carriers known to those of skill in the artare suitable for the present disclosure. Salts tend to be more solublein aqueous or other protonic solvents that are the corresponding freebase forms. In other cases, the preparation may be a lyophilized powderin 1 mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH rangeof 4.5 to 5.5, that is combined with buffer prior to use.

Thus, the compounds of the present disclosure may exist as salts, suchas with pharmaceutically acceptable acids. The present disclosureincludes such salts. Examples of such salts include hydrochlorides,hydrobromides, sulfates, methanesulfonates, nitrates, maleates,acetates, citrates, fumarates, tartrates (e.g., (+)-tartrates,(−)-tartrates, or mixtures thereof including racemic mixtures),succinates, benzoates, and salts with amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in theart.

An “adjuvant” (from Latin, adiuvare: to aid) is a pharmacological and/orimmunological agent that modifies the effect of other agents.

A “diluent” (also referred to as a filler, dilutant or thinner) is adiluting agent. Certain fluids are too viscous to be pumped easily ortoo dense to flow from one particular point to the other. This can beproblematic, because it might not be economically feasible to transportsuch fluids in this state. To ease this restricted movement, diluentsare added. This decreases the viscosity of the fluids, thereby alsodecreasing the pumping/transportation costs.

The terms “administration” or “administering” refer to the act ofproviding an agent of the current embodiments or pharmaceuticalcomposition including an agent of the current embodiments to theindividual in need of treatment.

By “co-administer” it is meant that a composition described herein isadministered at the same time, just prior to, or just after theadministration of additional therapies. The compound or the compositionof the disclosure can be administered alone or can be co-administered tothe patient. Co-administration is meant to include simultaneous orsequential administration of the compound individually or in combination(more than one compound or agent). The preparations can also becombined, when desired, with other active substances (e.g. to reducemetabolic degradation).

As used herein, “sequential administration” includes that theadministration of two agents (e.g., the compounds or compositionsdescribed herein) occurs separately on the same day or do not occur on asame day (e.g., occurs on consecutive days).

As used herein, “concurrent administration” includes overlapping induration at least in part. For example, when two agents (e.g., any ofthe agents or class of agents described herein that has bioactivity) areadministered concurrently, their administration occurs within a certaindesired time. The agents' administration may begin and end on the sameday. The administration of one agent can also precede the administrationof a second agent by day(s) as long as both agents are taken on the sameday at least once. Similarly, the administration of one agent can extendbeyond the administration of a second agent as long as both agents aretaken on the same day at least once. The bioactive agents/agents do nothave to be taken at the same time each day to include concurrentadministration.

As used herein, “intermittent administration includes the administrationof an agent for a period of time (which can be considered a “firstperiod of administration”), followed by a time during which the agent isnot taken or is taken at a lower maintenance dose (which can beconsidered “off-period”) followed by a period during which the agent isadministered again (which can be considered a “second period ofadministration”). Generally, during the second phase of administration,the dosage level of the agent will match that administered during thefirst period of administration but can be increased or decreased asmedically necessary.

As used herein, the term “administering” means oral administration,administration as a suppository, topical contact, intravenous,parenteral, intraperitoneal, intramuscular, intralesional, intrathecal,intranasal or subcutaneous administration, or the implantation of aslow-release device, e.g., a mini-osmotic pump, to a subject.Administration is by any route, including parenteral and transmucosal(e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, ortransdermal). Parenteral administration includes, e.g., intravenous,intramuscular, intra-arteriole, intradermal, subcutaneous,intraperitoneal, intraventricular, and. Other modes of delivery include,but are not limited to, the use of liposomal formulations, intravenousinfusion, transdermal patches, etc.

The compositions disclosed herein can be delivered transdermally, by atopical route, formulated as applicator sticks, solutions, suspensions,emulsions, gels, creams, ointments, pastes, jellies, paints, powders,and aerosols. Oral preparations include tablets, pills, powder, dragees,capsules, liquids, lozenges, cachets, gels, syrups, slurries,suspensions, etc., suitable for ingestion by the patient. Solid formpreparations include powders, tablets, pills, capsules, cachets,suppositories, and dispersible granules. Liquid form preparationsinclude solutions, suspensions, and emulsions, for example, water orwater/propylene glycol solutions. The compositions of the presentdisclosure may additionally include components to provide sustainedrelease and/or comfort. Such components include high molecular weight,anionic mucomimetic polymers, gelling polysaccharides and finely-divideddrug carrier substrates. These components are discussed in greaterdetail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760.The entire contents of these patents are incorporated herein byreference in their entirety for all purposes. The compositions disclosedherein can also be delivered as microspheres for slow release in thebody. For example, microspheres can be administered via intradermalinjection of drug-containing microspheres, which slowly releasesubcutaneously (see Rao, J. Bioniater Sci. Polym. Ed. 7:623-645, 1995;as biodegradable and injectable gel formulations (see, e.g., Gao Phann.Res. 12:857-863, 1995); or, as microspheres for oral administration(see, e.g., Eyles, J. Phann. Pharmacol. 49:669-674, 1997).

As used herein, an “effective amount” or “therapeutically effectiveamount” is that amount sufficient to affect a desired biological effect,such as beneficial results, including clinical results. As such, an“effective amount” depends upon the context in which it is beingapplied. An effective amount may vary according to factors known in theart, such as the disease state, age, sex, and weight of the individualbeing treated. Several divided doses may be administered daily or thedose may be proportionally reduced as indicated by the exigencies of thetherapeutic situation. In addition, the compositions/formulations ofthis disclosure can be administered as frequently as necessary toachieve a therapeutic amount.

Pharmaceutical compositions may include compositions wherein thetherapeutic drug (e.g., agents described herein, including embodimentsor examples) is contained in a therapeutically effective amount, i.e.,in an amount effective to achieve its intended purpose. The actualamount effective for a particular application will depend, inter alia,on the condition being treated. When administered in methods to treat adisease, such compositions will contain an amount of therapeutic drugeffective to achieve the desired result, e.g., modulating the activityof a target molecule, and/or reducing, eliminating, or slowing theprogression of disease symptoms.

The dosage and frequency (single or multiple doses) administered to amammal can vary depending upon a variety of factors, for example,whether the mammal suffers from another disease, and its route ofadministration; size, age, sex, health, body weight, body mass index,and diet of the recipient; nature and extent of symptoms of the diseasebeing treated, kind of concurrent treatment, complications from thedisease being treated or other health-related problems. Othertherapeutic regimens or agents can be used in conjunction with themethods and agents of this disclosure. Adjustment and manipulation ofestablished dosages (e.g., frequency and duration) are well within theability of those skilled in the art.

For any therapeutic agent described herein, the therapeuticallyeffective amount can be initially determined from cell culture assays.Target concentrations will be those concentrations of therapeuticdrug(s) that are capable of achieving the methods described herein, asmeasured using the methods described herein or known in the art.

As is well known in the art, therapeutically effective amounts for usein humans can also be determined from animal models. For example, a dosefor humans can be formulated to achieve a concentration that has beenfound to be effective in animals. The dosage in humans can be adjustedby monitoring agent's effectiveness and adjusting the dosage upwards ordownwards, as described above. Adjusting the dose to achieve maximalefficacy in humans based on the methods described above and othermethods is well within the capabilities of the ordinarily skilledartisan.

Dosages may be varied depending upon the requirements of the patient andthe therapeutic drug being employed. The dose administered to a patientshould be sufficient to effect a beneficial therapeutic response in thepatient over time. The size of the dose also will be determined by theexistence, nature, and extent of any adverse side-effects. Determinationof the proper dosage for a particular situation is within the skill ofthe practitioner. Generally, treatment is initiated with smaller dosageswhich are less than the optimum dose of the agent. Thereafter, thedosage is increased by small increments until the optimum effect undercircumstances is reached. Dosage amounts and intervals can be adjustedindividually to provide levels of the administered agent effective forthe particular clinical indication being treated. This will provide atherapeutic regimen that is commensurate with the severity of theindividual's disease state.

A weight percent of a component, unless specifically stated to thecontrary, is based on the total weight of the formulation or compositionin which the component is included.

“Excipient” is used herein to include any other agent that may becontained in or combined with a disclosed agent, in which the excipientis not a therapeutically or biologically active agent/agent. As such, anexcipient should be pharmaceutically or biologically acceptable orrelevant (for example, an excipient should generally be non-toxic to theindividual). “Excipient” includes a single such agent and is alsointended to include a plurality of excipients. For the purposes of thepresent disclosure the term “excipient” and “carrier” are usedinterchangeably in some embodiments of the present disclosure and saidterms are defined herein as, “ingredients which are used in the practiceof formulating a safe and effective pharmaceutical composition.”

The term “about” refers to any minimal alteration in the concentrationor amount of an agent that does not change the efficacy of the agent inpreparation of a formulation and in treatment of a disease or disorder.The term “about” with respect to concentration range of the agents(e.g., therapeutic/active agents) of the current disclosure also refersto any variation of a stated amount or range which would be an effectiveamount or range.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it is understood thatthe particular value forms another aspect. It is further understood thatthe endpoints of each of the ranges are significant both in relation tothe other endpoint, and independently of the other endpoint. It is alsounderstood that there are a number of values disclosed herein, and thateach value is also herein disclosed as “about” that particular value inaddition to the value itself. It is also understood that throughout theapplication, data are provided in a number of different formats and thatthis data represent endpoints and starting points and ranges for anycombination of the data points. For example, if a particular data point“10” and a particular data point “15” are disclosed, it is understoodthat greater than, greater than or equal to, less than, less than orequal to, and equal to 10 and 15 are considered disclosed as well asbetween 10 and 15. It is also understood that each unit between twoparticular units are also disclosed. For example, if 10 and 15 aredisclosed, then 11, 12, 13, and 14 are also disclosed.

Compound

In one aspect, provided herein is a compound including aphosphorothioated CpG oligodeoxynucleotide (CpG-ODN), conjugated to ananti-microRNA (anti-miR) or to a microRNA (miRNA)-mimic nucleic acidsequence (miRNA-mimic). In embodiments, the CpG-ODN is a 15 to 30 bases(nucleobases) long, single-stranded, partly or completelyphosphorothioated oligodeoxynucleotide.

In one aspect, provided herein is a compound including an anti-microRNA(anti-miR) sequence, where the anti-miR sequence includes one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more),phosphorothioate linkages and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, or more) chemically modified nucleotides.

In embodiments, the compound includes a nucleic acid sequence (CpG-ODN)having about 80%-100% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100%) sequence identity with at least a 15nucleobase continuous sequence of one of SEQ ID NOs: 1-14, conjugated toan anti-miR. In embodiments, the compound includes a nucleic acidsequence (CpG-ODN) having about 80%-100% (e.g., 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with one ofSEQ ID NOs: 1-14, conjugated to an anti-miR. In embodiments, providedherein is a compound including a nucleic acid sequence (CpG-ODN) havingabout 80%-100% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100%) sequence identity with at least a 15 nucleobasecontinuous sequence of one of SEQ ID NOs: 1-14, conjugated to amiRNA-mimic. In embodiments, the compound includes a nucleic acidsequence (CpG-ODN) having about 80%-100% (e.g., 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with one ofSEQ ID NOs: 1-14, conjugated to an miRNA-mimic. In embodiments, thenucleic acid sequence (CpG-ODN) is 15 to 30 (e.g., 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) bases long,single-stranded, partly or completely phosphorothioated oligonucleotide

In embodiments, the compound of the present disclosure includes anucleic acid sequence (CpG-ODN) having about 80-85%, about 85-90%, about90-95%, about 95%-100% sequence identity with at least a 15 nucleobasecontinuous sequence of one of SEQ ID NOs: 1-14, conjugated an anti-miRor a miRNA-mimic. In embodiments, the compound of the present disclosureincludes a nucleic acid sequence (CpG-ODN) having about 80-85%, about85-90%, about 90-95%, about 95%-100% sequence identity with one of SEQID NOs: 1-14, conjugated an anti-miR or a miRNA-mimic. In embodiments,the nucleic acid sequence (CpG-ODN) is a 15 to 30 (e.g., 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) bases long,single-stranded, partly or completely phosphorothioated oligonucleotide.

In embodiments, the anti-miR is anti-miR126. In embodiments, theanti-miR is anti-miR155. In embodiments, the anti-miR is anti-R125b. Inembodiments, the anti-miR is anti-miR146a. In embodiments, the anti-miRis anti-miR9. In embodiments, the anti-miR is anti-miR142. Inembodiments, the anti-miR is anti-miR10b. In embodiments, the anti-miRis anti-miR21. In embodiments, the anti-miR is anti-miR17. Inembodiments, the anti-miR is anti-miR92.

In embodiments, the miRNA-mimic is miR126-mimic. In embodiments, themiRNA-mimic is miR155-mimic. In embodiments, the miRNA-mimic isR125b-mimic. In embodiments, the miRNA-mimic is miR146a-mimic. Inembodiments, the miRNA-mimic is miR9-mimic. In embodiments, themiRNA-mimic is miR142-mimic. In embodiments, the miRNA-mimic ismiR10b-mimic. In embodiments, the miRNA-mimic is miR21-mimic. Inembodiments, the miRNA-mimic is miR17-mimic. In embodiments, themiRNA-mimic is miR92-mimic.

The nucleic acid sequences (CpG-ODN) of SEQ ID NOs: 1-14 are listed inTable 1.

TABLE 1 Compound and component sequences. SEQ SEQUENCE 5′-3′ ID NAME(* = phosphorothioate linkage) NO: CpG(A)- G*G*TGCATCGATGCAGG*G*G*G*G  1ODN GpC(A)- G*G*T GCA TGC ATG CAG G*G*G*G*G  2 ODN D19-PSG*G*T*G*C*A*T*C*G*A*T*G*C*A*G*G*G*G*G*G  3 CpG(B)-T*C*C*A*T*G*A*C*G*T*T*C*C*T*G*A*T*G*C*T  4 ODN ODN 1585G*G*GGTCAACGTTGAG*G*G*G*G*G or  5 G*GGGTCAACGTTGAG*G*G*G*G*G ODN 2216G*G*GGGACGA:TCGTCG*G*G*G*G*G or  6 G*GGGGACGA:TCGTCG*G*G*G*G*G ODN D19G*G*TGCATCGATGCAGG*G*G*G*G or  7 G*GTGCATCGATGCAGG*G*G*G*G ODN 2336G*G*G*GACGAC:GTCGTGG*G*G*G*G*G or  8 G*G*GGACGAC:GTCGTGG*G*G*G*G*GODN 1668 T*C*C*A*T*G*A*C*G*T*T*C*C*T*G*A*T*G*C*T  9 ODN 1826T*C*C*A*T*G*A*C*G*T*T*C*C*T*G*A*C*G*T*T 10 ODN 2006T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T 11 (ODN7909) ODN 2007T*C*G*T*C*G*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T 12 ODN 2395T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G 13 ODN M362T*C*G*T*C*G*T*C*G*T*T*C*G*A*A*C*G*A*C*G*T*T*G*A* 14 T

In embodiments, the anti-miRNA sequence is an anti-miR126, anti-miR142,anti-miR155, anti-miR125b, anti-miR146a, anti-miR9, anti-miR10b,anti-miR17, anti-miR18, anti-miR19, anti-miR20, anti-miR21, anti-miR22,anti-miR23, anti-miR24, anti-miR25, anti-miR26, anti-miR27, anti-miR28,anti-miR29, anti-miR30, anti-miR31, anti-miR32, anti-miR33, anti-miR34,anti-miR35, anti-miR36, anti-miR37, anti-miR38, anti-miR39, anti-miR40,anti-miR41, anti-miR42, anti-miR43, anti-miR44, anti-miR45, anti-miR46,anti-miR47, anti-miR48, anti-miR49, anti-miR50, anti-miR51, anti-miR52,anti-miR53, anti-miR54, anti-miR55, anti-miR56, anti-miR57, anti-miR58,anti-miR59, anti-miR60, anti-miR61, anti-miR62, anti-miR63, anti-miR64,anti-miR65, anti-miR66, anti-miR67, anti-miR68, anti-miR69, anti-miR70,anti-miR71, anti-miR72, anti-miR73, anti-miR74, anti-miR75, anti-miR76,anti-miR77, anti-miR78, anti-miR79, anti-miR80, anti-miR81, anti-miR82,anti-miR83, anti-miR84, anti-miR85, anti-miR86, anti-miR87, anti-miR88,anti-miR89, anti-miR90, anti-miR91, or anti-miR92 nucleic acid sequence.

In embodiment, the mimic of the compound of the present disclosure is amiR126-mimic, miR142-mimic, miR155-mimic, miR125b-mimic, miR146a-mimic,miR9-mimic, miR10b-mimic, miR17-mimic, miR18-mimic, miR19-mimic,miR20-mimic, miR21-mimic, miR22-mimic, miR23-mimic, miR24-mimic,miR25-mimic, miR26-mimic, miR27-mimic, miR28-mimic, miR29-mimic,miR30-mimic, miR31-mimic, miR32-mimic, miR33-mimic, miR34-mimic,miR35-mimic, miR36-mimic, miR37-mimic, miR38-mimic, miR39-mimic,miR40-mimic, miR41-mimic, miR42-mimic, miR43-mimic, miR44-mimic,miR45-mimic, miR46-mimic, miR47-mimic, miR48-mimic, miR49-mimic,miR50-mimic, miR51-mimic, miR52-mimic, miR53-mimic, miR54-mimic,miR55-mimic, miR56-mimic, miR57-mimic, miR58-mimic, miR59-mimic,miR60-mimic, miR61-mimic, miR62-mimic, miR63-mimic, miR64-mimic,miR65-mimic, miR66-mimic, miR67-mimic, miR68-mimic, miR69-mimic,miR70-mimic, miR71-mimic, miR72-mimic, miR73-mimic, miR74-mimic,miR75-mimic, miR76-mimic, miR77-mimic, miR78-mimic, miR79-mimic,miR80-mimic, miR81-mimic, miR82-mimic, miR83-mimic, miR84-mimic,miR85-mimic, miR86-mimic, miR87-mimic, miR88-mimic, miR89-mimic,miR90-mimic, miR91-mimic, or miR92-mimic nucleic acid sequence.

In embodiments, provided herein is a compound linking CpG-ODN to ananti-miR126, which has the sequence: 5′ G*G*T GCA TCG ATG CAGG*G*G* G*G(SEQ ID NO: 1) xxxxx CGC AUU AUU ACU CAC GGU ACG A (SEQ ID NO: 16) 3′and xxxxx indicates one or more linkers described herein. Inembodiments, provided herein is a compound linking CpG-ODN to ananti-miR126, which has the sequence: 5′ G*G*T GCA TCG ATG CAGG*G*G* G*G(SEQ ID NO: 1) xxxxx mCmGmC mAmUmU mAmUmU mAmCmU mCmAmC mGmGmU mAmCmG mA(SEQ ID NO: 17) 3′, where xxxxx indicates one or more linkers describedherein.

In embodiments, provided herein is a compound linking CpG-ODN to ananti-miR126, which has the sequence: 5′ G*GT GCA TGC ATG CAG G*G*G*G*G(SEQ ID NO: 2) xxxxx CGC AUU AUU ACU CAC GGU ACG A (SEQ ID NO: 16) 3′.In embodiments, provided herein is a compound linking CpG-ODN to ananti-miR126, which has the sequence: 5′ G*GT GCA TGC ATG CAG G*G*G*G*G(SEQ ID NO: 2) xxxxx mCmGmC mAmUmU mAmUmU mAmCmU mCmAmC mGmGmU mAmCmG mA(SEQ ID NO: 17) 3′, where xxxxx indicates one or more linkers describedherein.

In embodiments, provided herein is a compound linking CpG-ODN to ananti-miR126, which has the sequence: 5′G*G*T*G*C*A*T*C*G*A*T*G*C*A*G*G*G*G*G*G (SEQ ID NO: 3) xxxxx CGC AUU AUUACU CAC GGU ACG A (SEQ ID NO: 16) 3′. In embodiments, provided herein isa compound linking CpG-ODN to an anti-miR126, which has the sequence: 5′G*G*T*G*C*A*T*C*G*A*T*G*C*A*G*G*G*G*G*G (SEQ ID NO: 3) xxxxx mCmGmCmAmUmU mAmUmU mAmCmU mCmAmC mGmGmU mAmCmG mA (SEQ ID NO: 17) 3′, wherexxxxx indicates one or more linkers described herein. In embodiments,provided herein is a compound linking CpG-ODN to an anti-miR126, whichhas the sequence: 5′ G*G*T*G*C*A*T*C*G*A*T*G*C*A*G*G*G*G*G*G (SEQ ID NO:3) xxxxx mC*mG*mC* mA*mU*mU* mA*mU*mU* mA*mC*mU* mC*mA*mC* mG*mG*mU*mA*mC*mG*mA (SEQ ID NO: 48) 3′, where xxxxx indicates one or morelinkers described herein.

Exemplary miR126 mimic sequences are listed in Table 2.

TABLE 2 compounds including CpG-ODN linked to miR126 mimics CpG(D19)-PS + 3x 5′-G*G*TGCATCGATGCAGG SEQ ID Sense miR126 *G*G*G*G xxxxx rUrCrGNOS: unmodified rUr ArC rCrGrU rGrArG rUrArA 1, 18rUrArA rUrGrC rGrUrU-3′ CpG(D19)- PS + 3x 5′-G*G*TGCATCGATGCAGG SEQ IDSense miR126 2′F *G*G*G*G xxxxx rUrCrG NOS: FluororUrArC rCrGrU rGrArG rUrArA 1, 19 modified 2U rUrArA rUrGrC rGfUfU-3′at 3′ end CpG D19- PS + 3x 5′-G*G*TGCATCGATGCAGG SEQ ID Sense mir126 2′F*G*G*G*G xxxxx fUrAfC NO: All fCrGfU rGrArG fUrArA fUrArA 1, 20pyrimidines fUrGfC rGfUfU-3′ Fluoro modified Complementary5′-rCrGrC rArUrU rArUrU SEQ ID mir rArCrU rCrArC rGrGrU rArCrG NO: 21rA-3′ In the above sequences, xxxxx may be-(CH₂)_(n)-PO₄-[(CH₂)_(n)-PO₄]_(z)-(CH₂)_(n) *: phoshorothioation (Onenone-bridging oxygen replaced with sulfur)

In embodiments, the linker represented by “xxxxx” or the like describedherein (e.g. in Tables 2, 3, and 4 (infra)) is a bond, substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted alkylene, substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted heteroalkylene, substituted(e.g. substituted with a substituent group, size-limited substituentgroup or lower substituent group) or unsubstituted cycloalkylene,substituted (e.g. substituted with a substituent group, size-limitedsubstituent group or lower substituent group) or unsubstitutedheterocycloalkylene, substituted (e.g. substituted with a substituentgroup, size-limited substituent group or lower substituent group) orunsubstituted arylene, substituted (e.g. substituted with a substituentgroup, size-limited substituent group or lower substituent group) orunsubstituted cyclo-heteroalkylene or—(CH₂)_(n)—PO₄—[(CH₂)_(n)—PO₄]_(z)—(CH₂)_(n), in which the symbol n isan integer from 1 to 5 (e.g., 3) and the symbol z is an integer from 0to 50 (e.g. from 0 to 25, 0 to 10, or 0 to 5). In embodiments, n is 3and z is 0 to 5 or 1 to 5. In embodiments, n is 3 and z is 0 to 4 or 1to 4. In embodiments, n is 3 and z is 0 to 3 or 1 to 3. In embodiments,n is 3 and z is 3. 2′OMe (2′-O-Methylnucleoside; Hydroxyl in 2′-positionreplaced with 2′-OMethyl); PS is phoshorothioation. One none-bridgingoxygen replaced with sulfur; PS+3 represents three phosphates in thesequence modified, had one none-bridging oxygen replaced with sulfur;PS+5 represents five phosphates in the sequence modified, had onenone-bridging oxygen replaced with sulfur.

For example, as shown below, in embodiments, nucleobases in thephosphorothioated oligonucleotide of the present disclosure sequence mayinclude a phosphorothioate internucleotide linkage. A portion of such aphosphorothioated oligonucleotide is shown below.

The linker may have the structure below, where the linker connects withthe 3′ phosphate of the guanine on one end and the 5′ phosphate of thethymidine on the other end, and the nucleobases in the antisense partmay be modified with 2′OMe.

The above formula represents a portion of the CpG-ODN linked at the3′-OH end with a (CH₂)₃ linker (also referred to herein as the C₃linker), which links to the 5′-phosphate of the antisense RNA.

The linker may be a bond, substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) or unsubstituted alkylene, substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) or unsubstituted heteroalkylene, substituted (e.g. substitutedwith a substituent group, size-limited substituent group or lowersubstituent group) or unsubstituted cycloalkylene, substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted heterocycloalkylene,substituted (e.g. substituted with a substituent group, size-limitedsubstituent group or lower substituent group) or unsubstituted arylene,or substituted or unsubstituted heteroarylene.

In embodiments, the present disclosure includes a composition linkingphosphorothioated oligonucleotide of the present disclosure to ananti-miR142, which has the sequence: 5′ G*G*T GCA TCG ATG CAGG*G*G* G*G(SEQ ID NO: 1) xxxxx UCCAUAAAGUAGGAAACACUACA (SEQ ID NO: 22)₃′. ThemiR142 mimics are listed in Table 3:

TABLE 3 Compound and component sequences.SEQUENCE (* = phosphorothioate linkage), xxxxx maybe -(CH₂)_(n)-PO₄-[(CH₂)_(n)-PO₄]_(z)-(CH₂)_(n)) bonded tophosphate groups  at both ends except at the termini where terminalphosphates are optionally added and 5′x has an OH terminus and 3′ x has a -C⁶-NH₂ bonded to the final SEQ ID NAMEphosphate group, other linkages are phosphodiester. NOS: CpG D19-Sense5′-G*G*TGCATCGATGCAGG*G*G*G*G xxxxx 1, 23 miR142rUrGrU rArGrU rGrUrU rUrCrC rUrArC rUrUrU unmodified rArUrG rGrArUrU-3′CpG D12-Sense 5′-G*G*TGCATCGATGCAGG*G*G*G*G xxxxx 1, 24 miR42 fluororUrGrU rArGrU rGrUrU rUrCrC rUrArC rUrUrU modified 2U at 3′rArUrG rGrAfUfU-3′ end Complementary5′-rUrCrC rArUrA rArArG rUrArG rGrArA rArCrA 25 mir rCrUrA rCrA-3′

In embodiments, provided herein is a composition linking CpG-ODN to ananti-miR155 having the sequence: 5′ G*G*T GCA TCG ATG CAGG*G*G* G*G (SEQID NO: 1) xxxxx UGUUAAUGCUAAUAUGUAGGAG (SEQ ID NO: 26) 3′. Inembodiments, provided herein is a composition linking CpG-ODN to ananti-miR155 having the sequence: 5′ G*G*T GCA TCG ATG CAGG*G*G* G*G (SEQID NO: 1) xxxxx ACCCCTATCACAATTAGCATTAA (SEQ ID NO: 27) 3′, wherenucleotide T in SEQ ID NO: 27 can be substituted with nucleotide U. Inembodiments, a compound described herein includes a composition linkingCpG-ODN to an anti-miR155 having the sequence: 5′ G*G*T GCA TCG ATGCAGG*G*G* G*G (SEQ ID NO: 1) xxxxxmA*mC*mC*mC*mC*mU*mA*mU*mC*mA*mC*mA*mA*mU*mU*mA*mG*mC*mA*mU* mU*mA*mA(SEQ ID NO: 28) 3′. In embodiments, provided herein is a compositionlinking CpG-ODN to an anti-miR155 having the sequence: 5′T*G*C*T*G*C*T*T*T*T*G*T*G*C*T*T*T*T*G*T*G*C*T*T (SEQ ID NO: 11) xxxxxACCCCTATCACAATTAGCATTAA (SEQ ID NO: 27) 3′. In embodiments, providedherein is a composition linking CpG-ODN to an anti-miR155 having thesequence: 5′ T*G*C*T*G*C*T*T*T*T*G*T*G*C*T*T*T*T*G*T*G*C*T*T (SEQ ID NO:11) xxxxx mA*mC*mC*mC*mC*mU*mA*mU*mC*mA*mC*mA*mA*mU*mU*mA*mG*mC*mA*mU*mU*mA*mA (SEQ ID NO: 28) 3′. Sequences of the miR155 mimics are listedin Table 4:

TABLE 4 Compound and component sequences.SEQUENCE (* = phosphorothioate linkage), xxxxx maybe -(CH₂)_(n)-PO₄-[(CH₂)_(n)-PO₄]_(z)-(CH₂)_(n)) bonded tophosphate groups  at both ends except at the termini where terminalphosphates are optionally added and 5′x has an OH terminus and 3′ x has a -C⁶-NH₂ bonded to the final SEQ ID NAMEphosphate group, other linkages are phosphodiester. NOS: CpG D19-Sense5′-G*G*TGCATCGATGCAGG*G*G*G*G xxxxx 1, 29 miR155rCrUrC rCrUrA rCrArU rArUrU rArGrC rArUrU unmodified rArArC rArUrU-3′CpG D12-Sense 5′-G*G*TGCATCGATGCAGG*G*G*G*G xxxxx 1, 30 miR155 fluororCrUrC rCrUrA rCrArU rArUrU rArGrC rArUrU modified 2U at 3′rArArC rAfUfU-3′ end Complementary5′-rUrGrU rUrArA rUrGrC rUrArA rUrArU rGrUrA 31 mir rGrGrA rG-3′

In embodiments, a CpG(D19)-scrambled RNA of sequence 5′ G*G*T GCA TCGATG CAGG*G*G* G*G (SEQ ID NO: 1) xxxxxmGmUmAmGmAmAmCmCmGmUmAmCmUmCmGmUmCmAmCmUmUmA (SEQ ID NO: 32) 3′ is usedas a control ODN (included in FIG. 1 ).

In embodiments, provided herein is a composition linking CpG-ODN to ananti-miR125b having the sequence: 5′ G*G*T GCA TCG ATG CAGG*G*G* G*G(SEQ ID NO: 1) xxxxx TCACAAGTTAGGGTCTCAGGGA (SEQ ID NO: 33) 3′, wherenucleotide T can be substituted with nucleotide U in SEQ ID NO: 33. Inembodiments, provided herein is a composition linking CpG-ODN to ananti-miR125b having the sequence: 5′ G*G*T GCA TCG ATG CAGG*G*G* G*G(SEQ ID NO: 1) xxxxxmU*mC*mA*mC*mA*mA*mG*mU*mU*mA*mG*mG*mG*mU*mC*mU*mC*mA*mG*mG* mG*mA (SEQID NO: 34) 3′.

In embodiments, provided herein is a composition linking CpG-ODN to ananti-miR146a having the sequence: 5′ G*G*T GCA TCG ATG CAGG*G*G* G*G(SEQ ID NO: 1) xxxxx CCCATGGAATTCAGTTCTCA (SEQ ID NO: 35) 3′, wherenucleotide T can be substituted with nucleotide U in SEQ ID NO: 35. Inembodiments, provided herein is a composition linking CpG-ODN to ananti-miR125b having the sequence: 5′ G*G*T GCA TCG ATG CAGG*G*G* G*G(SEQ ID NO: 1) xxxxxmC*mC*mC*mA*mT*mG*mG*mA*mA*mT*mT*mC*mA*mG*mT*mT*mC*mT*mC*mA (SEQ ID NO:36) 3′. In embodiments, provided herein is a composition linking CpG-ODNto an anti-miR146a having the sequence: 5′ G*G*T GCA TGC ATG CAGG*G*G*G*G (SEQ ID NO: 2) xxxxx CCCATGGAATTCAGTTCTCA (SEQ ID NO: 35) 3′. Inembodiments, provided herein is a composition linking CpG-ODN to ananti-miR146a having the sequence: G*G*T GCA TGC ATG CAGG*G*G* G*G (SEQID NO: 2) xxxxxmC*mC*mC*mA*mT*mG*mG*mA*mA*mT*mT*mC*mA*mG*mT*mT*mC*mT*mC*mA (SEQ ID NO:36) 3′.

In embodiments, the compound including a nucleic acid sequence (CpG-ODN)is conjugated to an anti-miR or miRNA-mimic sequence, with one or morelinkers described herein.

In embodiments, the compound including a nucleic acid sequence (CpG-ODN)having about 80%-100% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100%) sequence identity with at least a 15nucleobase sequence of one of SEQ ID NOs: 1-14, is conjugated to ananti-miR or miRNA-mimic sequence, with one or more linkers describedherein.

In embodiments, the compound including a nucleic acid sequence (CpG-ODN)having about 80%-100% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100%) sequence identity with at least a 15nucleobase sequence of one of SEQ ID NOs: 1-14, is conjugated to anucleic acid sequence having about 80%-100% (e.g., 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity withat least a 15 nucleobase sequence of one of SEQ ID Nos 16-31, 33-36 and48, with one or more linkers described herein.

In embodiments, the compound including a nucleic acid sequence (CpG-ODN)having about 80%-100% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100%) sequence identity with at least a 15nucleobase sequence of one of SEQ ID NOs: 1-14, is conjugated to asecond nucleic acid sequence having about 80%-100% (e.g., 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identitywith at least a 15 nucleobase sequence of one of SEQ ID Nos 16-31, 33-36and 48, with one or more linkers described herein, where the secondnucleic acid sequence contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30 or more) phosphorothioate linkages.

In embodiments, the compound including a nucleic acid sequence (CpG-ODN)having about 80%-100% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100%) sequence identity with at least a 15nucleobase sequence of one of SEQ ID NOs: 1-14, is conjugated to asecond nucleic acid sequence having about 80%-100% (e.g., 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identitywith at least a 15 nucleobase sequence of one of SEQ ID Nos 16-31, 33-36and 48, with one or more linkers described herein, where the secondnucleic acid sequence contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30 or more) chemically modified nucleotides. Inembodiments, a chemical modification is selected from the groupconsisting of a 2′ 0-Methyl, 2′-deoxy-2′fluoro, 2′-deoxy, a universalbase, 5-C-methyl, an inverted deoxy abasic residue incorporation, and alocked nucleic acid.

In embodiments, the compound including a nucleic acid sequence (CpG-ODN)having about 80%-100% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100%) sequence identity with at least a 15nucleobase sequence of one of SEQ ID NOs: 1-14, is conjugated to asecond nucleic acid sequence having about 80%-100% (e.g., 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identitywith at least a 15 nucleobase sequence of one of SEQ ID Nos 16-31, 33-36and 48, with one or more linkers described herein, where the secondnucleic acid sequence contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30 or more) phosphorothioate linkages and one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) chemicallymodified nucleotides. In embodiments, a chemical modification isselected from the group consisting of a 2′ 0-Methyl, 2′-deoxy-2′fluoro,2′-deoxy, a universal base, 5-C-methyl, an inverted deoxy abasic residueincorporation, and a locked nucleic acid.

In embodiments, the compound including a nucleic acid sequence (CpG-ODN)selected from SEQ ID NOs: 1-14, is conjugated to an anti-miR ormiRNA-mimic sequence, with one or more linkers described herein.

In embodiments, the compound including a nucleic acid sequence (CpG-ODN)selected from one of SEQ ID NOs: 1-14, is conjugated to a nucleic acidsequence selected from SEQ ID Nos 16-31, 33-36 and 48, with one or morelinkers described herein.

In embodiments, the compound including a nucleic acid sequence (CpG-ODN)selected from one of SEQ ID NOs: 1-14, is conjugated to a second nucleicacid sequence selected from SEQ ID Nos 16-31, 33-36 and 48, with one ormore linkers described herein, where the second nucleic acid sequencecontains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 ormore) phosphorothioate linkages.

In embodiments, the compound including a nucleic acid sequence (CpG-ODN)selected from one of SEQ ID NOs: 1-14, is conjugated to a second nucleicacid sequence selected from SEQ ID Nos 16-31, 33-36 and 48, with one ormore linkers described herein, where the second nucleic acid sequencecontains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 ormore) chemically modified nucleotides. In embodiments, a chemicalmodification is selected from the group consisting of a 2′ 0-Methyl,2′-deoxy-2′fluoro, 2′-deoxy, a universal base, 5-C-methyl, an inverteddeoxy abasic residue incorporation, and a locked nucleic acid.

In embodiments, the compound including a nucleic acid sequence (CpG-ODN)selected from one of SEQ ID NOs: 1-14, is conjugated to a second nucleicacid sequence selected from SEQ ID Nos 16-31, 33-36 and 48, with one ormore linkers described herein, where the second nucleic acid sequencecontains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 ormore) phosphorothioate linkages and one or more (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30 or more) chemically modified nucleotides. Inembodiments, a chemical modification is selected from the groupconsisting of a 2′ 0-Methyl, 2′-deoxy-2′fluoro, 2′-deoxy, a universalbase, 5-C-methyl, an inverted deoxy abasic residue incorporation, and alocked nucleic acid.

In embodiments, the compound including a nucleic acid sequence (CpG-ODN)selected from one of SEQ ID NOs: 1-14, is conjugated to a second nucleicacid sequence selected from SEQ ID Nos 16-31, 33-36 and 48, with one ormore linkers described herein, where the second nucleic acid sequencecontains phosphorothioate linkages for all the internucleotide linkages.

In embodiments, the compound including a nucleic acid sequence (CpG-ODN)selected from one of SEQ ID NOs: 1-14, is conjugated to a second nucleicacid sequence selected from SEQ ID Nos 16-31, 33-36 and 48, with one ormore linkers described herein, where the second nucleic acid sequencecontains nucleotides that are all chemically modified (e.g., 2′0-Methyl).

In embodiments, the compound including a nucleic acid sequence (CpG-ODN)selected from one of SEQ ID NOs: 1-14, is conjugated to a second nucleicacid sequence selected from SEQ ID Nos 16-31, 33-36 and 48, with one ormore linkers described herein, where the second nucleic acid sequencecontains phosphorothioate linkages for all the internucleotide linkagesand contains nucleotides that are all chemically modified (e.g., 2′0-Methyl).

In embodiments, the compound including a nucleic acid sequence of ananti-miR or miRNA-mimic sequence, where the nucleic acid sequencecontains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 ormore) phosphorothioate linkages and/or one or more (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 or more) chemically modified nucleotides. Inembodiments, a chemical modification is selected from the groupconsisting of a 2′ 0-Methyl, 2′-deoxy-2′fluoro, 2′-deoxy, a universalbase, 5-C-methyl, an inverted deoxy abasic residue incorporation, and alocked nucleic acid.

In embodiments, the compound including a nucleic acid sequence of ananti-miR or miRNA-mimic sequence selected from SEQ ID Nos 16-31, 33-36and 48, where the nucleic acid sequence contains one or more (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30 or more) phosphorothioate linkagesand/or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more)chemically modified nucleotides. In embodiments, a chemical modificationis selected from the group consisting of a 2′ 0-Methyl,2′-deoxy-2′fluoro, 2′-deoxy, a universal base, 5-C-methyl, an inverteddeoxy abasic residue incorporation, and a locked nucleic acid.

In embodiments, the linker is a covalent linker (i.e. a linker thatcovalently attaches at least two (e.g. 2) portions of a compound). Inembodiments, the linker is or includes a substituted (e.g. substitutedwith a substituent group, size-limited substituent group or lowersubstituent group) or unsubstituted alkylene or heteroalkylene linker.In embodiments, the nucleic acid conjugated to anti-miRs and miRNAmimics includes more than one substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) or unsubstituted heteroalkylene linkers. Linkers may be addedduring the synthesis in sequence. In embodiments, heteroalkylene linkersare connected to each other with an intervening phosphate bond. Inembodiments, the covalent linker is a substituted (e.g. substituted witha substituent group, size-limited substituent group or lower substituentgroup) or unsubstituted heteroalkylene linker.

In embodiments, the linker is a substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) or unsubstituted heteroalkylene or substituted (e.g. substitutedwith a substituent group, size-limited substituent group or lowersubstituent group) or unsubstituted cyclo-heteroalkylene. A“cyclo-heteroalkylene,” as used herein is a heteroalkylene having a oneor more divalent cyclic moieties within the heteroalkylene chain. Thecyclic moiety may be a substituted (e.g. substituted with a substituentgroup, size-limited substituent group or lower substituent group) orunsubstituted cycloalklylene, substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted arylene or substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted heteroarylene. In embodiments,the cyclic moiety is a substituted (e.g. substituted with a substituentgroup, size-limited substituent group or lower substituent group) orunsubstituted ribose (e.g., a nucleoside). In embodiments, the cyclicmoiety serves as a branch point of the linker thereby forming a branchedlinker. The cyclic moiety branch point may be used to attach additionalfunctional moieties to the conjugates provided herein, such asdetectable moieties, drug moieties or biomolecule. As explained in moredetail below, the additional functional moieties may be connected usingclick chemistry techniques as known in the art.

In embodiments, the linker is or contains a moiety having the formula:

—(CH₂)_(n)—PO₄—[(CH₂)_(n)—PO₄]_(z)—(CH₂)_(n)—.

In the formula above, the symbol n is an integer from 1 to 5 (e.g., 3)and the symbol z is an integer from 0 to 50 (e.g. from 0 to 25, 0 to 10,or 0 to 5). In embodiments, n is 3 and z is 0 to 5 or 1 to 5. Inembodiments, n is 3 and z is 0 to 4 or 1 to 4. In embodiments, n is 3and z is 0 to 3 or 1 to 3. In embodiments, n is 3 and z is 3.

For example, the linker may have the structure below, where the linkerconnects with the 3′ phosphate of the guanine on one end and the 5′phosphate of the thymidine on the other end:

In embodiments, the guanidine above is connected to the nucleic acidsequence (CpG-ODN), and the thymidine is connected to an anti-miR ormiRNA-mimic sequence.

In embodiments, the linker may include a moiety selected from an azidegroup, a protected amino group, N-hydroxysuccinimide (NHS) group, and aprotected sulfhydryl group.

In embodiments, the linker may include a protected sulfhydryl group thatis conjugated to a moiety selected from the group consisting of divinylsulfone derivative, acryloyl derivative, and/or maleimido derivative. Inembodiments, the acryloyl derivative is acryloyl chloride.

In embodiments, linker may be conjugated to polyethylene glycol (PEG) orbisphosphonate moiety.

In embodiments, linker may include an unsubstituted C₃ heteroalkylene.

In embodiments, linker may include an unsubstituted C₆ to C₁₂heteroalkylene.

In embodiments, the linker may be substituted with a reactive group(e.g. a click chemistry reactive group) or a protected reactive group.The reactive group may be used to conjugate the CpG-ODN to an anti-miRor miRNA-mimic and/or to an additional functional moiety as describedherein, such as a detectable moiety or biomolecule (e.g. a targetingmoiety).

Thus, the linker may include further modification, conjugation, orattachment of additional moieties.

The reactive group used to conjugate the CpG-ODN to an anti-miR ormiRNA-mimic compound to an additional functional moiety may be anyapplicable reactive group useful in bioconjugate chemistry. SeeHermanson, Bioconjugate Techniques 1996, Academic Press, Inc., SanDiego.

In embodiments, the reactive group is a click chemistry reactive groups.Click chemistry refers to a group of reactions that are fast, simple touse, easy to purify, versatile, regiospecific, and give high productyields. Four different click reactions are possible: (1)Cycloadditions—these primarily refer to 1,3-dipolar cycloadditions, butalso include hetero-Diels-Alder cycloadditions; (2) Nucleophilicring-openings—these refer to the opening of strained heterocyclicelectrophiles, such as aziridines, epoxides, cyclic sulfates,aziridinium ions, episulfonium ions; (3) carbonyl chemistry of thenon-aldol type—examples include the formations of ureas, thioureas,hydrazones, oxime ethers, amides, aromatic heterocycles; (4) additionsof carbon-carbon multiple bonds—examples include epoxidations,aziridinations, dihydrooxylations, sulfenyl halide additions, nitrosylhalide additions, and certain Michael additions. In embodiments, theclick reaction used may be Cu′-catalyzed Huisgen 1,3-dipolarcycloaddition (HDC) of azides or terminal alkynes to form1,2,3-triazoles. In embodiments, the click reaction may be a copper-freereaction.

In embodiments, the click chemistry reactive group is or includes anazide groups, an alkene group, an amino groups, an N-hydroxysuccinimidegroup, a sulfhydryl group, a divinyl sulfone derivative, or a maleimidoderivative. Thus, in embodiments, the linker is substituted with areactive group (e.g. a click chemistry reactive group) or a protectedreactive group, including, for example, a protected amino group or aN-hydroxysuccinimide group, suitable for conjugation byN-hydroxysuccinimide (NETS) chemistry; a sulfhydryl group that may beconjugated with divinyl sulfone; a protected sulfhydryl group, which maybe conjugated with 1-alkyl-3-methylacryloyl (acryloyl) chloride oracryloyl derivatives; a protected sulfhydryl group, which may beconjugated with maleimido derivatives.

Provided below is a structural example of a cyclo-heteroalkylenebranched linker:

As shown above, a cyclo-heteroalkylene branched linker connects with the3′ phosphate of the guanine on one end and the 5′ phosphate of thethymidine on the other end. The moiety of the cyclo-heteroalkylenebranched linker is a branch point and is a 5-substituted thymidine. Thethymidine is substituted in position 5 with a reactive group containingan NHS moiety, which can serve as a reactive group to connect to anadditional functional moiety

Additional examples of compounds that can be used to serve as moietybranch points containing reactive functional groups and protectedreactive functional groups are provided below.

In embodiments, the linker branch point may be non-cyclic. An example ofa compound that can be used to serve as non-cyclic moiety branch pointwithin the linker that contains a reactive functional group andprotected reactive functional groups is provided below.

As set forth above, the reactive group may be used to conjugate theCpG-ODN to an anti-miR or miRNA mimic nucleic acid sequence and/or to anadditional functional moiety such as a detectable moiety, therapeuticmoiety (e.g., drug moiety), targeting moiety or biomolecule. Additionalfunctional moieties include a fluorescent label, a targeting compound(bone targeting bisphosphonates), a drug, or an antibody. Inembodiments, additional moiety is a chemically reactive moiety,detectable moiety, therapeutic moiety (e.g. anti-cancer agent oranti-viral agent), nucleic acid sequence, DNA sequence, or nucleic acidanalogs. In embodiments, the detectable moiety is a fluorescent dye,electron-dense reagent, enzyme, biotin, digoxigenin, paramagneticmolecule, paramagnetic nanoparticle, contrast agent, magnetic resonancecontrast agent, X-ray contrast agent, Gadolinium, radioisotope,radionuclide, fluorodeoxyglucose, gamma ray emitting radionuclide,positron-emitting radionuclide, biocolloid, microbubble, iodinatedcontrast agent, barium sulfate, thorium dioxide, gold, goldnanoparticle, gold nanoparticle aggregate, fluorophore, two-photonfluorophore, hapten, protein, or fluorescent moiety. In embodiments, anadditional moiety is a therapeutic moiety (e.g. anti-cancer agent oranti-viral agent).

In embodiments, the additional functional moiety is a substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted alkyl, substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted heteroalkyl, substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted cycloalkyl, substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted heterocycloalkyl, substituted(e.g. substituted with a substituent group, size-limited substituentgroup or lower substituent group) or unsubstituted aryl, or substituted(e.g. substituted with a substituent group, size-limited substituentgroup or lower substituent group) or unsubstituted heteroaryl. Inembodiments, the additional moiety is a substituted (e.g. substitutedwith a substituent group, size-limited substituent group or lowersubstituent group) or unsubstituted C₁-C₄₀ alkyl, substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted 2 to 40 membered heteroalkyl,substituted (e.g. substituted with a substituent group, size-limitedsubstituent group or lower substituent group) or unsubstituted C₃-C₈cycloalkyl, substituted or unsubstituted 3 to 8 memberedheterocycloalkyl, substituted or unsubstituted C₆-C₁₀ aryl, orsubstituted (e.g. substituted with a substituent group, size-limitedsubstituent group or lower substituent group) or unsubstituted 5 to 10membered heteroaryl. In embodiments, the additional moiety is asubstituted (e.g. substituted with a substituent group, size-limitedsubstituent group or lower substituent group) C₁-C₄₀ alkyl, substituted2 to 40 membered heteroalkyl, substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) C₃-C₈ cycloalkyl, substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) 3 to 8 membered heterocycloalkyl, substituted (e.g. substitutedwith a substituent group, size-limited substituent group or lowersubstituent group) C₆-C₁₀ aryl, or substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) 5 to 10 membered heteroaryl. In embodiments, the additionalfunctional moiety is an R¹-substituted C₁-C₄₀ alkyl, R¹-substituted 2 to40 membered heteroalkyl, R¹-substituted C₃-C₈ cycloalkyl, R¹-substituted3 to 8 membered heterocycloalkyl, R¹-substituted C₆-C₁₀ aryl, orR¹-substituted 5 to 10 membered heteroaryl. In embodiments, theadditional functional moiety is an R¹-substituted C₁-C₄₀ alkyl. Inembodiments, the additional functional moiety is an -(unsubstitutedC₁-C₄₀ alkylene)-R¹. In embodiments, the additional functional moiety isan -(unsubstituted linear C₁-C₄₀ alkylene)-R¹. In embodiments, theadditional functional moiety is an -(unsubstituted C₃-C₂₁ alkylene)-R¹.In embodiments, the additional functional moiety is an -(unsubstitutedC₃-C₁₈ alkylene)-R¹. In embodiments, the additional functional moiety isan -(unsubstituted linear C₃-C₁₅ alkylene)-R¹. In embodiments, theadditional functional moiety is an -(unsubstituted linear C₆-C₂₁alkylene)-R¹. In embodiments, the additional functional moiety is an-(unsubstituted linear C₉-C₂₁ alkylene)-R¹. In embodiments, theadditional functional moiety is an -(unsubstituted linear C₉-C₁₈alkylene)-R¹. In embodiments, the additional functional moiety is an-(unsubstituted linear C₉-C₁₅ alkylene)-R¹. In embodiments, theadditional functional moiety is an -(unsubstituted linear C₁₂-C₁₅alkylene)-R¹. In embodiments, the additional functional moiety is an-(unsubstituted linear C₁₂ alkylene)-R¹. In embodiments, the additionalfunctional moiety is an -(unsubstituted linear C₁₃ alkylene)-R¹. Inembodiments, the additional functional moiety is an -(unsubstitutedlinear C₁₄ alkylene)-R¹. In embodiments, the additional functionalmoiety is an -(unsubstituted linear C₁₅ alkylene)-R¹. In embodiments,the additional functional moiety is an R¹-substituted 2 to 40 memberedheteroalkyl. In embodiments, the additional functional moiety is an-(unsubstituted 2 to 40 membered heteroalkylene)-R¹. In embodiments, theadditional functional moiety is a -(substituted linear 2 to 40 memberedheteroalkylene)-R¹. In embodiments, the additional functional moiety isa -(substituted 5 to 40 membered heteroalkylene)-R¹. In embodiments, theadditional functional moiety is a -(substituted 10 to 40 memberedheteroalkylene)-R¹. In embodiments, the additional functional moiety isa -(substituted 15 to 40 membered heteroalkylene)-R¹. In embodiments,the additional functional moiety is a -(substituted 20 to 40 memberedheteroalkylene)-R¹. In embodiments, the additional functional moiety isa -(substituted 30 to 40 membered heteroalkylene)-R¹. In embodiments,the additional functional moiety is a -(substituted 2 to 35 memberedheteroalkylene)-R¹. In embodiments, the additional functional moiety isa -(substituted 2 to 30 membered heteroalkylene)-R¹. In embodiments, theadditional functional moiety is a -(substituted 2 to 25 memberedheteroalkylene)-R¹. In embodiments, the additional functional moiety isa -(substituted 2 to 20 membered heteroalkylene)-R¹. In embodiments, theadditional functional moiety is a -(substituted 2 to 10 memberedheteroalkylene)-R¹. In embodiments, the additional functional moiety isa -(substituted 2 to 50 membered heteroalkylene)-R¹. In embodiments, theadditional functional moiety is a -(substituted 2 to 60 memberedheteroalkylene)-R¹. In embodiments, the additional functional moiety isa substituted 2 to 40 membered heteroalkyl. In embodiments, theadditional functional moiety is a substituted 10 to 50 memberedheteroalkyl. In embodiments, the additional functional moiety is asubstituted 20 to 40 membered heteroalkyl. In embodiments, theadditional functional moiety is a substituted 25 to 40 memberedheteroalkyl. In embodiments, the additional functional moiety is asubstituted 30 to 40 membered heteroalkyl.

R¹ is a oxo, halogen, —CN, —CF₃, —NH₂, —OH, —SH, —N₃, substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted alkyl, substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted hetoeroalkyl, substituted(e.g. substituted with a substituent group, size-limited substituentgroup or lower substituent group) or unsubstituted substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted cycloalkyl, substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted heterocycloalkyl, substituted(e.g. substituted with a substituent group, size-limited substituentgroup or lower substituent group) or unsubstituted aryl or substitutedor unsubstituted heteroaryl. In embodiments, R¹ in an additionalfunctional moiety is a detectable moiety or a therapeutic moiety. Inembodiments, R¹ in an additional functional moiety is a detectablemoiety. In embodiments, the detectable moiety is a fluorescent dye,electron-dense reagent, enzyme, biotin, digoxigenin, paramagneticmolecule, paramagnetic nanoparticle, contrast agent, magnetic resonancecontrast agent, X-ray contrast agent, Gadolinium, radioisotope,radionuclide, fluorodeoxyglucose, gamma ray emitting radionuclide,positron-emitting radionuclide, biocolloid, microbubble, iodinatedcontrast agent, barium sulfate, thorium dioxide, gold, goldnanoparticle, gold nanoparticle aggregate, fluorophore, two-photonfluorophore, hapten, protein, or fluorescent moiety. In embodiments, R¹in an additional functional moiety is a therapeutic moiety (e.g.anti-cancer agent or anti-viral agent). In embodiments, R¹ in anadditional functional moiety is H. In embodiments, an additionalfunctional moiety is oxo. In embodiments, an additional functionalmoiety is oxygen. In embodiments, an additional functional moiety issulfur. In embodiments, an additional functional moiety is ═S.

In embodiments, the further linking substituent includes a substituted(e.g. substituted with a substituent group, size-limited substituentgroup or lower substituent group) or unsubstituted alkylene, substituted(e.g. substituted with a substituent group, size-limited substituentgroup or lower substituent group) or unsubstituted heteroalkylene,substituted (e.g. substituted with a substituent group, size-limitedsubstituent group or lower substituent group) or unsubstitutedcycloalkylene, substituted (e.g. substituted with a substituent group,size-limited substituent group or lower substituent group) orunsubstituted heterocycloalkylene, substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) or unsubstituted arylene or substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) or unsubstituted heteroarylene. The further linking substituentmay include a PEG moiety attached to the reactive group or additionalmoiety.

In embodiments, the linker includes an unsubstituted C₃ alkylene (e.g.as described above). In embodiments, the linker may be unsubstituted C₁₅alkylene. In embodiments, the linker includes an unsubstituted C₆ to C₁₆alkylene. In embodiments, the linker may be a substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted alkylene, substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted heteroalkylene, substituted(e.g. substituted with a substituent group, size-limited substituentgroup or lower substituent group) or unsubstituted cycloalkylene,substituted (e.g. substituted with a substituent group, size-limitedsubstituent group or lower substituent group) or unsubstitutedheterocycloalkylene, substituted (e.g. substituted with a substituentgroup, size-limited substituent group or lower substituent group) orunsubstituted arylene, or substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) or unsubstituted heteroarylene. In embodiments, the linker may bea substituted (e.g. substituted with a substituent group, size-limitedsubstituent group or lower substituent group) or unsubstituted C₁-C₄₀alkylene, substituted (e.g. substituted with a substituent group,size-limited substituent group or lower substituent group) orunsubstituted 2 to 40 membered heteroalkylene, substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted C₃-C₈ cycloalkylene,substituted (e.g. substituted with a substituent group, size-limitedsubstituent group or lower substituent group) or unsubstituted 3 to 8membered heterocycloalkylene, substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) or unsubstituted C₆-C₁₀ arylene, or substituted (e.g. substitutedwith a substituent group, size-limited substituent group or lowersubstituent group) or unsubstituted 5 to 10 membered heteroarylene. Inembodiments, the linker may be an unsubstituted C₁-C₄₀ alkylene,unsubstituted 2 to 40 membered heteroalkylene, unsubstituted C₃-C₈cycloalkylene, unsubstituted 3 to 8 membered heterocycloalkylene,unsubstituted C₆-C₁₀ arylene, or unsubstituted 5 to 10 memberedheteroarylene. In embodiments, the linker may be a substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) 2 to 40 membered heteroalkylene.

A linker may be a bond, nucleic acid sequence, two nucleic acidsequences, DNA sequence, two DNA sequences, nucleic acid analogsequence, substituted (e.g. substituted with a substituent group,size-limited substituent group or lower substituent group) orunsubstituted alkylene, substituted (e.g. substituted with a substituentgroup, size-limited substituent group or lower substituent group) orunsubstituted heteroalkylene, substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) or unsubstituted cycloalkylene, substituted (e.g. substitutedwith a substituent group, size-limited substituent group or lowersubstituent group) or unsubstituted heterocycloalkylene, substituted(e.g. substituted with a substituent group, size-limited substituentgroup or lower substituent group) or unsubstituted arylene, orsubstituted (e.g. substituted with a substituent group, size-limitedsubstituent group or lower substituent group) or unsubstitutedheteroarylene.

In embodiments, the linker is or contains a substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted alkylene, substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted heteroalkylene, substituted(e.g. substituted with a substituent group, size-limited substituentgroup or lower substituent group) or unsubstituted cycloalkylene,substituted (e.g. substituted with a substituent group, size-limitedsubstituent group or lower substituent group) or unsubstitutedheterocycloalkylene, substituted (e.g. substituted with a substituentgroup, size-limited substituent group or lower substituent group) orunsubstituted arylene, or substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) or unsubstituted heteroarylene. In embodiments, the linker is asubstituted (e.g. substituted with a substituent group, size-limitedsubstituent group or lower substituent group) or unsubstituted C₁-C₂₀alkylene, substituted (e.g. substituted with a substituent group,size-limited substituent group or lower substituent group) orunsubstituted 2 to 20 membered heteroalkylene, substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted C₃-C₈ cycloalkylene,substituted (e.g. substituted with a substituent group, size-limitedsubstituent group or lower substituent group) or unsubstituted 3 to 8membered heterocycloalkylene, substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) or unsubstituted C₆-C₁₀ arylene, or substituted (e.g. substitutedwith a substituent group, size-limited substituent group or lowersubstituent group) or unsubstituted 5 to 10 membered heteroarylene. Inembodiments, the linker is an unsubstituted C₁-C₂₀ alkylene,unsubstituted 2 to 20 membered heteroalkylene, unsubstituted C₃-C₈cycloalkylene, unsubstituted 3 to 8 membered heterocycloalkylene,unsubstituted C₆-C₁₀ arylene, or unsubstituted 5 to 10 memberedheteroarylene. In embodiments, the linker is an unsubstituted C₁-C₂₀alkylene. In embodiments, the linker is a substituted (e.g. substitutedwith a substituent group, size-limited substituent group or lowersubstituent group) or unsubstituted C₁-C₄₀ alkylene, substituted(e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted 2 to 40 memberedheteroalkylene, substituted (e.g. substituted with a substituent group,size-limited substituent group or lower substituent group) orunsubstituted C₃-C₈ cycloalkylene, substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) or unsubstituted 3 to 8 membered heterocycloalkylene, substituted(e.g. substituted with a substituent group, size-limited substituentgroup or lower substituent group) or unsubstituted C₆-C₁₀ arylene, orsubstituted (e.g. substituted with a substituent group, size-limitedsubstituent group or lower substituent group) or unsubstituted 5 to 10membered heteroarylene. In embodiments, the linker is a substituted(e.g. substituted with a substituent group, size-limited substituentgroup or lower substituent group) or unsubstituted C₁-C₄₀ alkylene. Inembodiments, the linker is a substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) or unsubstituted 2 to 40 membered heteroalkylene. In embodiments,the linker is a substituted (e.g. substituted with a substituent group,size-limited substituent group or lower substituent group) 2 to 40membered heteroalkylene. In embodiments, the linker includes alkylphosphates (e.g., propyl phosphates). In embodiments, the linker hasalkyl phosphates (e.g., propyl phosphates) bonded to the reminder of thecompound by phosphates at both ends. In embodiments, the linker has 1-5alkyl phosphates (e.g., propyl phosphates) bonded to the reminder of thecompound by phosphates at both ends. In embodiments, the linker has 1-4alkyl phosphates (e.g., propyl phosphates) bonded to the reminder of thecompound by phosphates at both ends. In embodiments, the linker has 4alkyl phosphates (e.g., propyl phosphates) bonded to the reminder of thecompound by phosphates at both ends. A person having ordinary skill inthe art will recognize that a linker having alkyl phosphates that isbonded to the remainder of the compound by phosphates on both ends willhave one more phosphate than alkylene groups (e.g., a linker having 4alkyl phosphates that is bonded to the reminder of the compound byphosphates at both ends will have five phosphates and four alkyl groupswith alternating phosphate groups and alkyl groups).

In embodiments, anti-miR and miRNA mimics may include modifications suchas 2′ 0-Methyl, 2′-deoxy-2′fluoro, 2′-deoxy, a universal base,5-C-methyl, an inverted deoxy abasic residue incorporation, or a lockednucleic acid, or any combination(s) thereof. In embodiments, theanti-miR and miRNA mimics may have a modification positioned at theterminal nucleobase of the anti-miR and miRNA mimics. In embodiments,the anti-miR and miRNA mimics may not have a modification positioned atthe terminal nucleobase of the anti-miR and miRNA mimics. Inembodiments, the modification of the anti-miR and miRNA mimics protectsthe compound against serum-derived nucleases (e.g. is nucleaseresistant).

In embodiments, the (CpG-ODN) conjugated to an anti-miR or miRNA mimichas a terminal moiety. A terminal moiety is a chemically reactivemoiety, detectable moiety, therapeutic moiety (e.g. anti-cancer agent oranti-viral agent), nucleic acid sequence, DNA sequence, substituted(e.g. substituted with a substituent group, size-limited substituentgroup or lower substituent group) or unsubstituted alkyl, substituted(e.g. substituted with a substituent group, size-limited substituentgroup or lower substituent group) or unsubstituted heteroalkyl,substituted (e.g. substituted with a substituent group, size-limitedsubstituent group or lower substituent group) or unsubstitutedcycloalkyl, substituted (e.g. substituted with a substituent group,size-limited substituent group or lower substituent group) orunsubstituted heterocycloalkyl, substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) or unsubstituted aryl, or substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) or unsubstituted heteroaryl.

In embodiments, a terminal moiety is a chemically reactive moiety,detectable moiety, therapeutic moiety (e.g. anti-cancer agent oranti-viral agent), nucleic acid sequence, DNA sequence, nucleic acidanalogs, R¹-substituted or unsubstituted alkyl, R¹-substituted orunsubstituted heteroalkyl, R¹-substituted or unsubstituted cycloalkyl,R¹-substituted or unsubstituted heterocycloalkyl, R¹-substituted orunsubstituted aryl, or R¹-substituted or unsubstituted heteroaryl.

In embodiments, a CpG-ODN nucleic acid sequence conjugated to ananti-miR or miRNA-mimic conjugates includes a terminal moiety, whereinthe terminal moiety is a detectable moiety. In embodiments, the CpG-ODNconjugated to an anti-miR or miRNA-mimic includes a terminal detectablemoiety such as, a fluorescent dye, electron-dense reagent, enzyme,biotin, digoxigenin, paramagnetic molecule, paramagnetic nanoparticle,contrast agent, magnetic resonance contrast agent, X-ray contrast agent,Gadolinium, radioisotope, radionuclide, fluorodeoxyglucose, gamma rayemitting radionuclide, positron-emitting radionuclide, biocolloid,microbubble, iodinated contrast agent, barium sulfate, thorium dioxide,gold, gold nanoparticle, gold nanoparticle aggregate, fluorophore,two-photon fluorophore, hapten, protein, or fluorescent moiety. Inembodiments, the CpG-ODN conjugated to an anti-miR or miRNA-mimicincludes a terminal moiety, which is a therapeutic moiety (e.g.,anti-cancer agent or anti-viral agent).

In embodiments, the CpG-ODN conjugated to an anti-miR or miRNA-mimicincludes a terminal moiety, which is a hydrogen, oxo, halogen, —CN,—CF₃, —NH₂, —OH, —SH, —N₃, substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) or unsubstituted alkyl, substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) or unsubstituted heteroalkyl, substituted (e.g. substituted witha substituent group, size-limited substituent group or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) or unsubstituted heterocycloalkyl, substituted (e.g. substitutedwith a substituent group, size-limited substituent group or lowersubstituent group) or unsubstituted aryl, or substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted heteroaryl. In embodiments,the CpG-ODN conjugated to an anti-miR or miRNA-mimic includes a terminalmoiety, which is a substituted (e.g. substituted with a substituentgroup, size-limited substituent group or lower substituent group) orunsubstituted C₁-C₄₀ alkyl, substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) or unsubstituted 2 to 40 membered heteroalkyl, substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) or unsubstituted C₃-C₈ cycloalkyl, substituted(e.g. substituted with a substituent group, size-limited substituentgroup or lower substituent group) or unsubstituted 3 to 8 memberedheterocycloalkyl, substituted (e.g. substituted with a substituentgroup, size-limited substituent group or lower substituent group) orunsubstituted C₆-C₁₀ aryl, or substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) or unsubstituted 5 to 10 membered heteroaryl. In embodiments, theCpG-ODN conjugated to an anti-miR or miRNA-mimic includes a terminalmoiety, which is a substituted (e.g. substituted with a substituentgroup, size-limited substituent group or lower substituent group) C₁-C₄₀alkyl, substituted (e.g. substituted with a substituent group,size-limited substituent group or lower substituent group) 2 to 40membered heteroalkyl, substituted (e.g. substituted with a substituentgroup, size-limited substituent group or lower substituent group) C₃-C₈cycloalkyl, substituted (e.g. substituted with a substituent group,size-limited substituent group or lower substituent group) 3 to 8membered heterocycloalkyl, substituted (e.g. substituted with asubstituent group, size-limited substituent group or lower substituentgroup) C₆-C₁₀ aryl, or substituted (e.g. substituted with a substituentgroup, size-limited substituent group or lower substituent group) 5 to10 membered heteroaryl. In embodiments, the terminal moiety is anR¹-substituted C₁-C₄₀ alkyl, R¹-substituted 2 to 40 memberedheteroalkyl, R¹-substituted C₃-C₈ cycloalkyl, R¹-substituted 3 to 8membered heterocycloalkyl, R¹-substituted C₆-C₁₀ aryl, or R¹-substituted5 to 10 membered heteroaryl. In embodiments, the terminal moiety is anR¹-substituted C₁-C₄₀ alkyl. In embodiments, the terminal moiety is an-(unsubstituted C₁-C₄₀ alkylene)-R¹. In embodiments, the terminal moietyis an -(unsubstituted linear C₁-C₄₀ alkylene)-R¹. In embodiments, theterminal moiety is an -(unsubstituted C₃-C₂₁ alkylene)-R¹. Inembodiments, the terminal moiety is an -(unsubstituted C₃-C₁₈alkylene)-R¹. In embodiments, the terminal moiety is an -(unsubstitutedlinear C₃-C₁₅ alkylene)-R¹. In embodiments, the terminal moiety is an-(unsubstituted linear C₆-C₂₁ alkylene)-R¹. In embodiments, the terminalmoiety is an -(unsubstituted linear C₉-C₂₁ alkylene)-R¹. In embodiments,the terminal moiety is an -(unsubstituted linear C₉-C₁₈ alkylene)-R¹. Inembodiments, the terminal moiety is an -(unsubstituted linear C₉-C₁₅alkylene)-R¹. In embodiments, the terminal moiety is an -(unsubstitutedlinear C₁₂ alkylene)-R¹. In embodiments, the terminal moiety is an-(unsubstituted linear C₁₃ alkylene)-R¹. In embodiments, the terminalmoiety is an -(unsubstituted linear C₁₄ alkylene)-R¹. In embodiments,the terminal moiety is an -(unsubstituted linear C₁₅ alkylene)-R¹. Inembodiments, the terminal moiety is an R¹-substituted 2 to 40 memberedheteroalkyl. In embodiments, the terminal moiety is an -(unsubstituted 2to 40 membered heteroalkylene)-R¹. In embodiments, the terminal moietyis a -(substituted linear 2 to 40 membered heteroalkylene)-R¹. Inembodiments, the terminal moiety is a -(substituted 5 to 40 memberedheteroalkylene)-R¹. In embodiments, the terminal moiety is a-(substituted 10 to 40 membered heteroalkylene)-R¹. In embodiments, theterminal moiety is a -(substituted 15 to 40 membered heteroalkylene)-R¹.In embodiments, the terminal moiety is a -(substituted 20 to 40 memberedheteroalkylene)-R¹. In embodiments, the terminal moiety is a-(substituted 30 to 40 membered heteroalkylene)-R¹. In embodiments, theterminal moiety is a -(substituted 2 to 35 membered heteroalkylene)-R¹.In embodiments, the terminal moiety is a -(substituted 2 to 30 memberedheteroalkylene)-R¹. In embodiments, the terminal moiety is a-(substituted 2 to 25 membered heteroalkylene)-R¹. In embodiments, theterminal moiety is a -(substituted 2 to 20 membered heteroalkylene)-R¹.In embodiments, the terminal moiety is a -(substituted 2 to 10 memberedheteroalkylene)-R¹. In embodiments, the terminal moiety is a-(substituted 2 to 50 membered heteroalkylene)-R¹. In embodiments, theterminal moiety is a -(substituted 2 to 60 membered heteroalkylene)-R¹.

In embodiments, the CpG-ODN conjugated to an anti-miR or miRNA-mimicincludes a terminal moiety, which is a substituted (e.g. substitutedwith a substituent group, size-limited substituent group or lowersubstituent group) 2 to 40 membered heteroalkyl. In embodiments, theCpG-ODN conjugated to an anti-miR or miRNA-mimic includes a terminalmoiety, which is a substituted (e.g. substituted with a substituentgroup, size-limited substituent group or lower substituent group) 10 to50 membered heteroalkyl. In embodiments, the CpG-ODN conjugated to ananti-miR or miRNA-mimic includes a terminal moiety, which is asubstituted (e.g. substituted with a substituent group, size-limitedsubstituent group or lower substituent group) 20 to 40 memberedheteroalkyl. In embodiments, the CpG-ODN conjugated to an anti-miR ormiRNA-mimic includes a terminal moiety, which is a substituted (e.g.substituted with a substituent group, size-limited substituent group orlower substituent group) 25 to 40 membered heteroalkyl. In embodiments,the CpG-ODN conjugated to an anti-miR or miRNA-mimic includes a terminalmoiety, which is a substituted (e.g. substituted with a substituentgroup, size-limited substituent group or lower substituent group) 30 to40 membered heteroalkyl.

In embodiments, the CpG-ODN conjugated to an anti-miR or miRNA-mimicincludes a terminal moiety with a R¹ group, in which R¹ is a detectablemoiety or a therapeutic moiety. In embodiments, R¹ in the CpG-ODNconjugated to an anti-miR or miRNA-mimic includes a terminal moiety,which is a detectable moiety. In embodiments, R¹ in the CpG-ODNconjugated to an anti-miR or miRNA-mimic includes a detectable moiety,which is a fluorescent dye, electron-dense reagent, enzyme, biotin,digoxigenin, paramagnetic molecule, paramagnetic nanoparticle, contrastagent, magnetic resonance contrast agent, X-ray contrast agent,Gadolinium, radioisotope, radionuclide, fluorodeoxyglucose, gamma rayemitting radionuclide, positron-emitting radionuclide, biocolloid,microbubble, iodinated contrast agent, barium sulfate, thorium dioxide,gold, gold nanoparticle, gold nanoparticle aggregate, fluorophore,two-photon fluorophore, hapten, protein, or fluorescent moiety. Inembodiments, R¹ in the CpG-ODN conjugated to an anti-miR or miRNA-mimicincludes a terminal moiety, which is a therapeutic moiety (e.g.,anti-cancer agent or anti-viral agent). In embodiments, R¹ in theCpG-ODN conjugated to an anti-miR or miRNA-mimic includes a terminalmoiety, which is H. In embodiments, R¹ in the CpG-ODN conjugated to ananti-miR or miRNA-mimic includes an oxo as a terminal moiety. Inembodiments, R¹ in the CpG-ODN conjugated to an anti-miR or miRNA-mimicincludes oxygen as a terminal moiety. In embodiments, R¹ in the CpG-ODNconjugated to an anti-miR or miRNA-mimic includes sulfur as a terminalmoiety. In embodiments, R¹ in the CpG-ODN conjugated to an anti-miR ormiRNA-mimic includes ═S as a terminal moiety.

In embodiments, the CpG-ODN nucleic acid sequence of the compoundincludes unmethylated CpG motif (e.g., a CpG nucleic acid sequence or aGpC nucleic acid sequence). In embodiments, the CpG-ODN nucleic acidsequence includes a Class A CpG nucleic acid sequence, a Class B CpGnucleic acid sequence, or a Class C CpG nucleic acid sequence.

In embodiments, the compound includes CpG-ODN, in which C and G arenucleotides connected by a phosphodiester internucleotide linkage. Inembodiments, the compound includes CpG, wherein C and G are nucleotidesconnected by a phosphodiester derivative internucleotide linkage. Inembodiments, the CpG motif is unmethylated. In embodiments, C and G areconnected as 5′C-G 3′. In embodiments, C and G are connected as 5′G-C3′.

In embodiments, a Toll-like receptor (TLR)-binding DNA substituent is aClass A CpG oligodeoxynucleotide (ODN). In embodiments, a TLR-bindingDNA substituent is a Class B CpG oligodeoxynucleotide (ODN). Inembodiments, a TLR-binding DNA substituent is a Class C CpGoligodeoxynucleotide (ODN). In embodiments, a TLR-binding DNAsubstituent (e.g., TLR9-binding DNA substituent) consists ofdeoxyribonucleic acids with A, G, C, or T bases and phosphodiesterlinkages and/or phosphodiester derivative linkages (e.g.,phosphorothioate linkage(s)).

In embodiments, the compound binds an endosomal TLR. In embodiments, thecompound preferentially binds an endosomal TLR over other TLR. Inembodiments, the compound specifically binds an endosomal TLR. Inembodiments, the compound binds TLR3. In embodiments, the compoundpreferentially binds TLR3 over other TLR. In embodiments, the compoundspecifically binds TLR3. In embodiments, the compound binds TLR7. Inembodiments, the compound preferentially binds TLR7 over other TLR. Inembodiments, the compound specifically binds TLR7. In embodiments, thecompound binds TLR8. In embodiments, the compound preferentially bindsTLR8 over other TLR. In embodiments, the compound specifically bindsTLR8. In embodiments, the compound binds TLR9. In embodiments, thecompound preferentially binds TLR9 over other TLR. In embodiments, thecompound specifically binds TLR9. In embodiments, the compound includesCpG, wherein C and G are nucleotides connected by a phosphodiesterinternucleotide linkage or phosphodiester derivative internucleotidelinkage.

In embodiments, the TLR-binding DNA substituent is a Class A CpGoligodeoxynucleotide (ODN). In embodiments, the TLR-binding DNAsubstituent is a Class B CpG oligodeoxynucleotide (ODN). In embodiments,the TLR-binding DNA substituent is a Class C CpG oligodeoxynucleotide(ODN). In embodiments, the TLR-binding DNA substituent is ODN 1585, ODN2216, ODN D19, or ODN 2336. In embodiments, the TLR-binding DNAsubstituent is ODN 1668, ODN 1826, ODN 2006, or ODN 2007. Inembodiments, the TLR-binding DNA substituent is ODN 2395 or ODN M362. Inembodiments, the TLR-binding DNA substituent is a derivative of ODN1585, ODN 2216, ODN D19, ODN 2336, ODN 1668, ODN 1826, ODN 2006, ODN2007, ODN 2395 or ODN M362. In embodiments, a derivative of ODN 1585,ODN 2216, ODN D19, ODN 2336, ODN 1668, ODN 1826, ODN 2006, ODN 2007, ODN2395 or ODN M362 includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10) nucleotide substitutions (e.g., A, C, G, or T substituted with adifferent nucleotide). In embodiments, a derivative of ODN 1585, ODN2216, ODN D19, ODN 2336, ODN 1668, ODN 1826, ODN 2006, ODN 2007, ODN2395 or ODN M362 includes one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10) internucleotide linkage replacements (e.g., phosphodiesterreplaced with a phosphodiester derivative or a phosphodiester derivativereplaced with a phosphodiester). In embodiments, a derivative of ODN1585, ODN 2216, ODN D19, ODN 2336, ODN 1668, ODN 1826, ODN 2006, ODN2007, ODN 2395 or ODN M362 includes one or more (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100) nucleotide deletions. In embodiments, a derivative ofODN 1585, ODN 2216, ODN D19, ODN 2336, ODN 1668, ODN 1826, ODN 2006, ODN2007, ODN 2395 or ODN M362 includes one or more (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, or 10) nucleotide additions.

In embodiments, the compound includes a phosphodiester derivativelinkage (e.g., phosphoramidate, phosphorodiamidate, phosphorothioate,phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates,phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boronphosphonate, or O-methylphosphoroamidite linkages). In embodiments, thecompound includes a plurality of phosphodiester derivative linkages(e.g., phosphoramidate, phosphorodiamidate, phosphorothioate,phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates,phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boronphosphonate, O-methylphosphoroamidite linkages, or combinationsthereof). In embodiments, the compound includes a phosphodiesterderivative linkage (e.g., phosphoramidate, phosphorodiamidate,phosphorothioate, phosphorodithioate, phosphonocarboxylic acids,phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid,methyl phosphonate, boron phosphonate, or O-methylphosphoroamiditelinkages) in the TLR9-binding DNA substituent. In embodiments, thecompound includes a phosphodiester derivative linkage (e.g.,phosphoramidate, phosphorodiamidate, phosphorothioate,phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates,phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boronphosphonate, or O-methylphosphoroamidite linkages) in the TLR-bindingnucleic acid (e.g., endosomal TLR-, TLR3-, TLR7-, TLR8-, or TLR9-bindingnucleic acid) substituent.

In embodiments, the phosphodiester derivative linkage in the compoundmay be phosphoramidate linkage, phosphorodiamidate linkage,phosphorothioate linkage, phosphorodithioate linkage,phosphonocarboxylic acid linkage, phosphonocarboxylate linkage,phosphonoacetic acid linkage, phosphonoformic acid linkage, methylphosphonate linkage, boron phosphonate linkage, orO-methylphosphoroamidite linkage.

In embodiments, one or more of the nucleic acid internucleotide linkagesin the compound is a phosphodiester derivative linkage (e.g.,phosphoramidate, phosphorodiamidate, phosphorothioate,phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates,phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boronphosphonate, or O-methylphosphoroamidite linkages), (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or all internucleotide linkages in the compound arephosphodiester derivative linkages (e.g., phosphoramidate,phosphorodiamidate, phosphorothioate, phosphorodithioate,phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid,phosphonoformic acid, methyl phosphonate, boron phosphonate,O-methylphosphoroamidite linkages, or combinations thereof)).

In embodiments, provided herein is a compound linking a CpG-ODN to ananti-miR targeting miR-126 or an miRNA-mimic of miR-142.

In embodiments, provided herein is a compound linking a CpG-ODN to ananti-miR targeting miR-155.

In embodiments, provided herein is a compound linking a CpG-ODN to ananti-miR targeting miR-125b.

In embodiments, provided herein is a compound linking a CpG-ODN to ananti-miR targeting miR-146a or an miRNA-mimic of miR-146a.

In embodiments, the CpG-ODN conjugated to an anti-miR or miRNA-mimic ispresent in the cytoplasm (and in the cell nucleus).

Pharmaceutical Composition

In one aspect, provided herein is pharmaceutical compositions includinga pharmaceutically acceptable excipient and a compound disclosed herein.In embodiments, the composition includes a second therapeutic agent. Inembodiments, the second therapeutic agent is a anti-tumor or anti-canceragent, anti-angiogenic agent, cytotoxic agent, cytostatic agent,anti-inflammatory agent, analgesic, anti-infective agent, growthinhibitory agent, immunogenic agent, immunomodulatory agent, or achemokine. In embodiments, an anti-tumor or anti-cancer agent in thepharmaceutical composition of the present disclosure is a cell deathpromoting agent.

In embodiments, the second therapeutic agent in the pharmaceuticalcomposition of the present disclosure includes a second therapeuticagent, for example ctinomycin D/Dactinomycin, Bleomycin, Daunorubicin,Doxorubicin, Doxorubicin (pegylated liposomal), Epirubicin, Idarubicin,Mitomycin, Mitoxantrone, Etoposide, Docetaxel, Irinotecan, Paclitaxel,Topotecan, Vinblastine, Vincristine, Vinorelbine, Carboplatin,Cisplantin, Oxaliplatin, Alemtuzamab, BCG, Bevacizumab, Cetuximab,Denosumab, Erlotinib, Gefitinib, Imatinib, Interferon, Ipilimumab,Lapatinib, Monomethyl auristatin E (MMEA), Mertansine (DM1), Rituximab,Sunitinib, Sorafenib, Temsirolimus, Trastuzumab, or any combination(s)thereof.

In embodiments, the present disclosure includes compositions of acombination of a compound of the present disclosure with one or moreadditional anti-cancer therapies, e.g., an anti-VEGF antibody, oranti-STAT agents.

In embodiments of any of the methods and uses, the disclosure includestreating cancer, by administering effective amounts of a compound of thepresent disclosure and a chemotherapeutic agents to a subject diagnosedwith cancer. A variety of chemotherapeutic agents may be used in thecombined treatment methods and uses of the present disclosure. Inembodiments, the chemotherapeutic agent may be temolozolomide. Inembodiments, the chemotherapeutic agent may be administeredconcommitantly with radiotherapy.

In one example, the combined treatment may involve administration whichincludes simultaneous administration, using separate formulations or asingle pharmaceutical formulation, and consecutive administration ineither order, where there may be a time period when both (or all) activeagents simultaneously exert their biological activities. Preparation anddosing schedules for such chemotherapeutic agents may be used accordingto manufacturers' instructions or as determined empirically by theskilled practitioner. Preparation and dosing schedules for chemotherapyare also described in Chemotherapy Service Ed., M. C. Perry, Williams &Wilkins, Baltimore, Md. (1992). The chemotherapeutic agent may precede,or follow administration of a compound or composition of the presentdisclosure or may be given simultaneously therewith.

In embodiments of any of the methods and uses, other therapeutic agentsuseful for combination tumor therapy with a compound of the presentdisclosure include antagonist of other factors that are involved intumor growth, such as VEGF, EGFR, ErbB3, ErbB4, STAT or TNF. Sometimes,it may be beneficial to also administer one or more cytokines to thesubject. In embodiments, a compound or composition of the presentdisclosure is co-administered with a growth inhibitory agent. Forexample, the growth inhibitory agent may be administered first, followedby the compound or composition of the present disclosure. However,simultaneous administration or administration of a compound orcomposition of the present disclosure first may be possible. Suitabledosages for the growth inhibitory agent are those presently used and maybe lowered due to the combined action (synergy) of the growth inhibitoryagent and a compound of the present disclosure.

The composition herein may also contain more than one active compound asnecessary for the particular indication being treated, e.g., those withcomplementary activities that do not adversely affect each other. Forexample, it may be desirable to further provide agents which bind toEGFR, VEGF (e.g., an antibody which binds a different epitope or sameepitope on VEGF), VEGFR, or ErbB2 in the one formulation. Such moleculesmay be suitably present in combination in amounts that are effective forthe purpose intended.

In embodiments of the methods and uses, other therapeutic agents usefulfor combination cancer therapy with a compound or composition of thepresent disclosure include other anti-angiogenic agents. Manyanti-angiogenic agents have been identified and are known in the arts,including those listed by Carmeliet and Jain (2000). In embodiments, acompound or composition of the present disclosure is used in combinationwith another miR antagonist, neutralizing antibodies against miRcomplex, low molecule weight inhibitors of miR, and any combinationsthereof.

The present disclosure includes compositions with an effective dose of acompound of the present disclosure. The effective dose may be betweenabout 0.001 mg/kg to about 100 mg/kg of the agent (e.g., 0.001, 0.005,0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100 mg/kg).

In embodiments, an effective dose of a compound including an anti-miR,e.g., anti-miR126, of the present disclosure is administered to asubject in need thereof for treating a disease (e.g., a cancer, anautoimmune disease or an infectious disease). The anti-miR suppressesexpression/activity of a miR (e.g., miR126) in a cell, and induces thecancer cell (e.g., leukemia stem cell (LSC)) to undergo cell cycle, andrender the cancer cell (e.g., LS) more sensitive to chemotherapy.

In embodiments, an effective dose of a compound including a miR-mimic,e.g., miR142-mimic, of the present disclosure is administered to asubject in need thereof for treating a disease (e.g., a cancer, anautoimmune disease or an infectious disease). The miR-mimic blockscancer, e.g., leukemia, development.

The compound may be administered to a subject in need thereof, at a dosebetween about 0.001 mg/kg to about 0.01 mg/kg of the compound, betweenabout 0.01 mg/kg to about 0.1 mg/kg of the compound, between about 0.1mg/kg to about 1.0 mg/kg of the compound, between about 1.0 mg/kg toabout 5.0 mg/kg of the compound, between about 5.0 mg/kg to about 10mg/kg of the compound, between about 10 mg/kg to about 15 mg/kg of thecompound, between about 15 mg/kg to about 20 mg/kg of the compound,between about 20 mg/kg to about 25 mg/kg of the compound, between about25 mg/kg to about 30 mg/kg of the compound, between about 30 mg/kg toabout 35 mg/kg of the compound, between about 35 mg/kg to about 40 mg/kgof the compound, between about 40 mg/kg to about 45 mg/kg of thecompound, between about 45 mg/kg to about 50 mg/kg of the compound,between about 50 mg/kg to about 55 mg/kg of the compound, between about55 mg/kg to about 60 mg/kg of the compound, between about 60 mg/kg toabout 65 mg/kg of the compound, between about 65 mg/kg to about 70 mg/kgof the compound, between about 70 mg/kg to about 75 mg/kg of thecompound, between about 75 mg/kg to about 80 mg/kg of the compound,between about 80 mg/kg to about 85 mg/kg of the compound, between about85 mg/kg to about 90 mg/kg of the compound, between about 90 mg/kg toabout 95 mg/kg of the compound, or between about 95 mg/kg to about 100mg/kg of the compound.

In embodiments, the present disclosure includes compositions with aneffective dose of a compound of the present disclosure in which thecompound may be between about 0.1% to about 20% w/v of the composition.

For example, the effective dose of a compound disclosed herein may bebetween about 0.001%-about 0.01%, between about 0.01%-about 0.1%,between about 0.1%-about 1.0%, between about 1.0%-about 2.0%, betweenabout 2.0%-about 3.0%, between about 3.0%-about 4.0%, between about4.0%-about 5.0%, between about 5.0%-about 6.0%, between about 6.0%-about7.0%, between about 7.0%-about 8.0%, between about 8.0%-about 9.0%,between about 9.0%-about 10%, between about 10%-about 11%, between about11%-about 12%, between about 12%-about 13%, between about 13%-about 14%,between about 14%-about 15%, between about 15%-about 16%, between about16%-about 17%, between about 17%-about 18%, between about 18%-about 19%,or between about 19%-about 20% w/v of the composition.

Methods of Treatment or Use

Provided herein is a method of treating a disease in a subject in needthereof, the method including administering to the subject an effectiveamount of a compound or the pharmaceutical composition including acompound disclosed herein. In embodiments, the disease is a cancer, anautoimmune disease or an infectious disease.

In embodiments, the cancer may be a hematopoietic cell cancer. Inembodiments, the cancer is not a hematopoietic cell cancer. Inembodiments, the cancer is myeloma or acute myeloid leukemia or chronicmyeloid leukemia. In embodiments, the cancer is prostate cancer, breastcancer, glioblastoma, ovarian cancer, lung cancer, head and neck cancer,esophageal cancer, skin cancer, melanoma, brain cancer, colorectalcancer, leukemia, lymphoma, or myeloma.

In embodiments, the autoimmune disease is rheumatoid arthritis,psoriasis, systemic lupus erythematosus (SLE), type II diabetes,non-alcoholic fatty liver disease, non-alcoholic steatohepatitis,multiple sclerosis (MS), Parkinson's disease (PD, Alzheimer's disease(AD) or inflammatory bowel disease (IBD).

In embodiments, the infectious disease is tuberculosis, influenza,Ebola, HIV, HPV infection or hepatitis.

In embodiments, the compound or the composition is administered to thesubject by intravenous, parenteral, subcutaneous, intramuscular,transdermal, intraperitoneal, intranasal, aerosol, oral, or topicaladministration. In embodiments, the treatment is dose-dependent of thecompound or composition. In embodiments, about 0.001 mg/kg to about 100mg/kg of the compound is administered to the subject. All digits andvarious ranges within this range are also implied.

Provided herein is a method of suppressing miR, e.g., miR126, in a cell,the method including contacting the cell with an effective amount of acompound or a pharmaceutical composition of a compound disclosed herein.Provided herein is a method of inhibiting cell growth includingcontacting the cell with an effective amount of a compound or apharmaceutical composition of a compound disclosed herein.

In embodiments, the cell is a cancer cell. In embodiments, the cell isan acute myeloid lymphoid (AML) cell or a chronic myeloid leukemia cell(CML). In embodiments, the AML cell is from the bone marrow. Inembodiments, the cell is a cultured cell in vitro; the cell is in situin a host; the cell is in a cultured tissue ex vivo. In embodiments, thecontacting step is free of viral transduction. In embodiments, thecontacting step is free of viral transduction and the cell is contactedwith a compound of the present disclosure or a pharmaceuticalcomposition including a compound of the present disclosure. Inembodiments the cell is contacted with about 1 nanomole to about 100nanomoles (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 nm) of the compound. Alldigits and various ranges within this range are also implied.

In embodiments, the heteroalkylene linker allow for furthermodification, conjugation, or attachment of additional moieties aftercompletion of the synthesis and while the oligonucleotide is stillattached to the support.

In embodiments, provided herein is a CpG-ODN conjugated to an anti-miRor miRNA-mimic with a substituted (e.g. substituted with a substituentgroup, size-limited substituent group or lower substituent group)heteroalkylene linker, which may allow further modification,conjugation, or attachment during synthesis and while theoligonucleotide is attached to a support.

In embodiments, the substituted (e.g. substituted with a substituentgroup, size-limited substituent group or lower substituent group)heteroalkylene linker is modified, conjugated, or attached tosubstituents. A modification may include the conversion of the originalsubstituent into a different substituent. For example, a bromo-alkanesubstituent may be converted into an azido-alkane. Conjugation mayresult in bonding of two large moieties together. For example, an NHSderivative may be conjugated with PEG-NH₂. A peptide may also beconjugated with an oligonucleotide or an antibody may be conjugated toan oligonucleotide. Attachment may result in bonding of the smallmolecule to a large molecule. For example, NETS-ester of biotin might beattached to the amino derivative of an oligo.

In embodiments, provided herein is a compound having CpG-ODN conjugatedto an anti-miR or miRNA-mimic with linkers multiple different linkers,multiple identical linkers, or a substitution of linkers selected fromthe following groups:

-   -   Fmoc amino-modifier C6 dT (introduction of the amino group)        which can be further used for functionalization, by reacting        with NHS ester and divinyl sulfone and its analogues.

-   -   S-Bz-thiol-modifier C6-dT (introduction of sulfuhydryl group),        which can be further used for functionalization by reacting with        divinyl sulfone and acrylic analogues.

-   -   Amino-modifier Serinol Phosphoramidite (introduction of the        amino group) which can be further be used for functionalization,        by reacting with NHS ester and divinyl sulfone and its        analogues.

-   -   DBCO-dT (introduction of alkyne, copper free Click Chemistry)        which can be further used for functionalization with        azido-reactants

-   -   DBCO-sulfo-NHS Ester (introduction of the of alkyne, copper free        Click Chemistry, by reacting with the amino groups)

Examples Example 1: Uptake of CpG-Anti-miR126 Inhibitor in Normal, CordBlood, AML and CML CD34+ Cells

Normal, cord blood, AML and CML CD34+ cells were cultured withCpG-anti-miR126 inhibitor-Cy3 or anti-miR126 inhibitor-Cy3 for 16 hours,and the uptake was measured by Cy3 expression in the cells by flowcytometry (FIGS. 2A-2D). All tested cells internalizedCpG-anti-miR126-Cy3 but not the anti-miR126-Cy3 with high efficiency.

Example 2: Uptake and Expression of CpG-Anti-miR126 Inhibitor in AML andCML Cell Lines

CML (K562) (FIG. 3A) and AML cell lines (KG1A, MV4-11, Molm13, NB4, OCIand HL60) (FIGS. 3B-3G) were cultured with anti-miR126 inhibitor-Cy3(Cy3-200 nM) alone, human CD45 (Ab-200 nM) or Transferrin (TF-200nM)-conjugated nanoparticles (NP) containing anti-miR126-Cy3 orunformulated CpG-anti-miR126 inhibitor-Cy3 in two concentrations (200 nMand 500 nM) for 4 hours, the uptake was analyzed by measuring Cy3expression in these cells by flow cytometry. The myeloid cell-specificCpG-anti-miR126 conjugate was quickly and dose-dependently internalizedby a variety of human AML and CML cell lines in vitro. The level ofCpG-anti-miR126 internalization in absence of any transfection regentsexceeded that of all other oligonucleotides including NP-formulatedanti-miR126 inhibitor

miRNA 126 expression in AML and CML lines was evaluated. CML (K562)(FIG. 4A) and AML cell lines (KG1A, MV4-11, Molm13, NB4, OCI and HL60)(FIGS. 4B-4G) were cultured with anti-miR126 inhibitor-Cy3 (Cy3 ctrl,200 nM), human CD45 (Ab-NP, 200 nM) or Transferrin (TF-NP, 200nM)-conjugated NP containing anti-miR-126-Cy3, or CpG-anti-miR126inhibitor-Cy3 (CpG-200 nM and 500 nM) for 24 hours, then miR126 andRNU44 (control) expression in these cells were analyzed by Q-RT-PCR. Themyeloid cell-specific CpG-anti-miR-126 conjugate (500 nM) leds to themost effective downregulation of miRNA126 in all the tested human AMLand CML cell lines in vitro, with more than 50% reduction of targetmiRNA expression in HL60, K562, MV4-11, MOLM13 and OCI cells

Example 3: miRNA126 Expression in NL/CB, AML and CML CD34+ Cells

Normal, cord blood, AML and CML CD34+ cells were cultured withCpG-scramble RNA (500 nM) and CpG-anti-miR126 inhibitor-Cy3 (500 nM) for24 hours, and then miR126 and RNU44 (ctrl) expression in these cellswere analyzed by Q-RT-PCR (FIG. 5 ). The miR126 expression levels werenormalized to RNU44 and calculated by using the comparative 2^(−ΔΔCt)method. The myeloid cell-specific CpG-anti-miR-126 conjugate (500 nM)was quickly internalized by a variety of primary patients' AML and CMLCD34+ cells in vitro and significant reduction of miR126 expression(60%-90% reduction) was seen in these cells.

Example 4: CML CD34+CD38− Primitive Progenitors Showed Higher miR126Expression than CD34+CD38+ Committed Progenitors

CML CD34+, CD34+CD38+ committed and CD34⁺CD38− primitive progenitorswere sorted and then miRNA126 and RNU44 (control) expression in thesecells were analyzed by Q-RT-PCR. The miR126 expression levels werenormalized to RNU44 and calculated by using the comparative 2^(−ΔΔCt)method (FIG. 6 ). CML CD34+CD38− primitive progenitors showed highermiR126 expression than CD34+CD38+ committed progenitors.

Example 5: Increased Apoptosis of CML CD34+, CD34+CD38+ Committed andCD34+CD38− Primitive Progenitors Treated with CpG-Anti-miR126 Inhibitorand NIL

CML CD34+, CD34+CD38+ committed and CD34+CD38− primitive progenitorswere cultured with CpG-scramble RNA (500 nM), CpG-anti-miR126 inhibitor(500 nM), CpG-scramble RNA (500 nM)+Nilotinib (NIL, 5 uM), andCpG-anti-miR126 inhibitor (500 nM)+NIL (5 uM) for 72 hours and then cellcycling and apoptosis were analyzed by EDU/DAPi and Annexin V/DAPistaining. More than 90% reduction of miR126 expression was seen inCpG-anti-miR126 inhibitor treated cells (FIG. 7 ). Increased apoptosisof CML CD34+, CD34+CD38+ committed and CD34+CD38− primitive progenitorstreated with CpG-anti-miR126 inhibitor and NIL was observed, comparedwith the cells treated with CpG-scramble and NIL (FIG. 8 ). Increasedcell cycling was seen in normal and CML CD34+CD38− primitive progenitorstreated with CpG-anti-miR126 inhibitor (FIG. 9 ).

Moreover, incubation with CpG-anti-miR-126 inhibitor (500 nM) resultedin increased cell-cycle entry of long term hematopoietic stem cells(LTHSC, Lin-Sca-1+c-kit+Fit3-CD150+CD48−) from normal and CML mouse,measured by EDU/DAPi staining (FIG. 10 ), compared with the CpG-Scr.CpG-anti-miR126 inhibitor combined with NIL also resulted insignificantly increased apoptosis (FIG. 11A) and significant reductionof cell growth (FIG. 11B) of LSC compared with CpG-SCR+NIL treatment.

Example 6: Silencing of miR126 by CpG-Anti-miR-126 Inhibitor Combinedwith Arabinose-c and Doxo Significantly Increased Apoptosis of LSCCompared to Each Treatment Alone

Silencing of miR-126 in human AML CD34+ cells by CpG-anti-miR-126inhibitor (500 nM) combined with Ara-c and Doxo significantly increasedcell cycling (FIG. 12A) and apoptosis (FIG. 12B) compared with Ara-c andDoxo alone, resulting in reduction of cell growth (FIG. 12C).

Example 7: In Vivo Effect of CpG-Anti-miR126 on Growth of Primary AMLand CML LSC and Therapeutic Response

SCLtTA/BCR-ABL mice were treated with CpG-miR-126 inhibitor (5 mg/kg,every other day, iv injection), SCR (5 mg/kg, every other day, ivinjection), NIL(50 mg/kg, daily by garage)+SCR, NIL+miR-126 inhibitorfor 3 weeks and then the remaining CML cells in the PB, BM and spleenwere analyzed. Reduced CML white blood cells in the PB of the micetreated with the NIL+miR126 inhibitor were seen (FIG. 13A), comparedwith the mice treated with NIL+SCR. Reduced spleen weight of the micetreated with the NIL+miR126 inhibitor were seen (FIG. 13B), comparedwith the mice treated with NIL+SCR. Reduced CML cells, CML LSK and CMLLTHSC were observed in the BM and spleen of the mice treated withNIL+miR126 inhibitor compared with the mice treated with NIL+SCR (FIGS.13C-D, FIGS. 14A-D). The effect of CpG-anti-miR-126 on growth of primaryAML LSC and therapeutic response in vivo is still ongoing. Theseobservations indicate that blocking of miR-126 by a myeloidcell-specific CpG-anti-miR-126 ODN inhibitor, among other compounds ofthe present disclosure, is very effective, and therefore represents anovel therapeutic method targeting miRNA in leukemia and other types ofcancer disclosed herein.

Example 8: Uptake and Inhibitory Effect of CpG-Anti-miRNAs

Sequences of exemplary compounds used in the studies are:

CpG-anti-miR155: 5′G*G*T GCA TCG ATG CAGG*G*G* G*G (SEQ ID NO: 1) xxxxxmA*mC*mC*mC*mC*mU*mA*mU*mC*mA*mC*mA*mA*mU*mU*mA*mG*mC*mA*mU*mU*mA*mA (SEQ ID NO: 28) 3′; CpG-anti-miR125b:5′ G*G*T GCA TCG ATG CAGG*G*G* G*G (SEQ ID NO: 1) xxXXXmU*mC*mA*mC*mA*mA*mG*mU*mU*mA*mG*mG*mG*mU*mC*mU*mC*mA*mG*mG*mG*mA (SEQ ID NO: 34) 3′; and CpG-anti-miR146a:5′ G*G*T GCA TCG ATG CAGG*G*G* G*G (SEQ ID NO: 1) xxXXXmC*mC*mC*mA*mU*mG*mG*mA*mA*mU*mU*mC*mA*mG*mU*mU*mC*mU*mC*mA(SEQ ID NO: 36) 3′,where * indicates a phosphorothioate linkage, mN indicates a 2′OMemodified nucleotide, and x indicates a linker described herein.

CpG-anti-miRNAs were Cy3-labeled to detect the intracellular uptake bytarget cells using flow cytometry. Human immune cells were incubatedwith indicated concentrations of CpG-anti-miR146a, CpG-anti-miR155 orCpG-anti-miR125b and uptake of these compounds was observed in the cells(FIGS. 16A-16B, 17A-17F, 18A-18F, 19A-19H). Treatment of these compoundsreduces the corresponding miR expression in human and mouse myeloidcells (FIGS. 17A-17F, 18A-18F, 19A-19H).

Effective knockdown of miR-126 with miR-126 inhibitors conjugated withCpG, GpC and PS, and effective over-expression with miR-126 mimics inK562 and MV4-11 cells. K562 and MV4-11 cells were treated with miR-126inhibitors conjugated with CpG, GpC, PS (FIGS. 26A-26B) or miR-126mimics (615, 616 and 617) (FIGS. 26C-26D) (500 nM) for 24 hours, andmiR-126 expression was measured in these cells. We showed here that CpGmotif can be omitted in the targeting ODN sequence. GpC and completelyPS-modified oligo also succeeds in blocking miR126. Incubation withmiR-126 mimics, especially GM617, significantly increased miR-126expression in K562 and MV4-11 cells. Just like CpG-miR-126 inhibitorwhich is very effective in reducing miR-126 in cells, we also designedmiR-126 mimics, which are very effective in increasing miR-126 levels incells without using any transduction reagents.

Example 9: Effect of CpG-Anti-miRNAs on Downstream Targets

CpG-anti-miRs regulate downstream targets of miR155, miR125b, andmiR146a (FIGS. 20A-20C). Mouse RAW264.7 or human MV4-11 cells wereincubated with 250 nM or 500 nM of CpGanti-miR155, CpG-anti-miR125b, orCpG-anti-miR146a, or 500 nM of CpG-scramble for 48 h, then the celllysates were collected and electrophoresed and immunoblotted byantibodies against SHIP1 (miR155 target) (FIG. 20A), IRF4 (miR125btarget) (FIG. 20B), or IRAK1 (miR146a target) (FIG. 20C). The bandintensities were normalized against (3-actin and quantified. Foldinduction over the control protein levels are indicated below the blot.FIG. 20D) MV4-11 cells were incubated with 500 nM of CpG-anti-miR155,CpG-antimiR125b, CpG-anti-miR146a, or CpG-scramble for 24 h, then celllysates were collected and electrophoresed and immunoblotted to detectactivated caspase 3 indicating induction of apoptosis. SHIP1 and IRAK1are both upregulated after the treatment of CpG-anti-miRs.

Example 10: Comparing the Inhibition Effects of CpG-Anti-miR andGpC-Anti-miR

CpG-anti-miR155, GpC-anti-miR155, CpG-anti-miR146a, and GpC-anti-miR146atreatment reduces miR155 or miR-146a expression in RAW264.7 (FIGS. 21A,21C) and A20 cells (FIGS. 21B, 21D). The cells were incubated with 100nM CpG-anti-miRs or GpC-anti-miRs for 18 hrs. FIGS. 21E-21H)CpG-anti-miRs and GpC-anti-miRs treatment regulates downstream targetsof miR155 and miR146a. RAW264.7 (FIGS. 21E,21G) or A20 cells (FIGS. 21F,21H) were incubated with 500 nM of CpG-anti-miR155, GpC-anti-miR155, orCpG-anti-miR146a, GpC-anti-miR146a for 48 hrs, then the cell lysateswere collected and immunoblotted using antibodies against SHIP1 (miR155target) or IRAK1 (miR146a target). These results demonstrate that bothCpG and GpC nucleic acid sequences are effective in the compoundsdescribed herein.

Example 11: CpG-miR146a Mimic Attenuates LPS Induced InflammatorySignaling

CpG-miR146a mimic increases miR-146a expression in cultured CMM leukemia(FIG. 22A) and A20 lymphoma cells (FIG. 22B). Cells were incubated with100 nM CpG-miR146a mimic for 18 h. FIG. 22C: CpG-miR146a mimic inhibitsIRAK1 expression, a downstream target of miR146a. A20 cells wereincubated with 500 nM of CpG-miR146a mimic or LPS (used as a positivecontrol) for 48 h, then the cell lysates were collected andimmunoblotted using IRAK1-specific antibodies. FIGS. 22D-22E) RAW-Bluecells, expressing NF-KB-responsive reporter gene, were treated with 500nM of CpG-miR146a mimic for 24 h and then with 1 pg/ml LPS for another24 h. Culture medium was collected and analyzed for NF-KB activity usingthe Quanti-Blue assay kit (FIG. 22D) for IL-6 levels in media usingELISA (FIG. 22E).

Example 12: Effective In Vitro and In Vivo Uptake and Gene SilencingEffects of the CpG-miR-126 Inhibitor

Uptake test measured by flow cytometric analysis at 4 hours (FIG. 23A)and 24 hours (FIG. 23B) after addition of CpG-miR-126 inhibitor-Cy3,Ab-NPs or TF-NPs containing miR-126 inhibitor-Cy3, or naked miR-126inhibitor-Cy3 in K562 cells. The experiment was replicated twice.miR-126 expression in K562 was measured by Q-RT-PCR at 24 hours (n=3)(FIG. 23C). Uptake in HUVEC (FIG. 23D), human normal (FIG. 23E) and CML(FIG. 23F) CD34⁺CD38⁻ cells at 4 hours after addition of CpG-miR-126inhibitor-Cy3 (500 nM) was measured by flow cytometry. miR-126expression in HUVEC (FIG. 23G), normal (FIG. 23H) and CML (FIG. 23I)CD34⁺CD38⁻ cells treated with CpG-miR-126 inhibitor (500 nM) for 24hours is shown (n=4). One of the two cell cycling experiments in normal(FIG. 23J) and CML (FIG. 23K) CD34⁺CD38⁻ cells treated with CpG-miR-126inhibitor (500 nM) by EDU staining is shown.

Murine CML BM, LTHSC and EC cells were treated with CpG-miR-126inhibitor-Cy3 (500 nM) for 4 hours and then Cy3+ cells were detected byflow cytometry (FIG. 25A). The cells were also collected at 24 hours andmiR-126 expression was determined by Q-RT-PCR (FIG. 25B). Cell cyclingwas measured by EDU staining at 72 hours after addition of CpG-miR-126inhibitor in CML BM LTHSC. One of the two representative plots is shownin (FIG. 25C). CML mice were treated with CpG-miR-126 inhibitor-Cy3 withone dose (5 mg/kg, iv injection) and Cy3 uptake in BM, LTHSC and EC wasmeasured at 16 hours after treatment by flow cytometry (FIG. 25D).Normal and CML mice were also treated with CpG-miR-126 inhibitor (5mg/kg/day, iv, daily) for 3 days and BM, LTHSC and EC from femurs weresorted and miR-126 expression was determined by Q-RT-PCR (FIGS.25E-25F). Wild type B6 mice were treated with CpG-scrRNA (scrRNA) orCpG-miR-126 inhibitor (Inhibitor) (5 mg/kg/day, iv injection) for 3weeks and BM cells were collected and analyzed. Red cell (RBC, FIG.25G), WBC (FIG. 25H), PLT (FIG. 25I), BM mononuclear cell (FIG. 25J),LTHSC (FIG. 25K) and EC (FIG. 25L) numbers are shown. BM cells (CD45.2)from the treated normal mice were transplanted into CD45.1 congenicrecipient mice and the donor cell engraftment in blood (FIG. 25M) and inBM and spleen at 16 weeks (FIG. 25N) and the donor LTHSC number in BM at16 weeks (FIG. 25O) was monitored. Results shown represent mean±SEM.*p<0.05, **p<0.01, ***p<0.001. Abbreviations: EC (endothelial cells);PLT (platelet).

Example 13: Combination of a Compound Described Herein and AnotherTherapeutic Agent

Knockdown of miR-126 by CpG-miR-126 inhibitor enhances elimination ofmouse CML LSC in combination with NIL in vivo. BM cells fromSCL-tTA/BCR-ABL mice (CD45.2) were transplanted into congenic B6 mice(CD45.1, n=40) to generate a cohort of mice with CML-like disease.Following confirmation of CML development at 4 weeks aftertransplantation, mice were randomly divided into 4 groups (n=10 each)and treated with CpG-miR-126 inhibitor (5 mg/kg i.v.4 times a week),CpG-scrRNA (5 mg/kg, i.v. 4 times a week), CpG-miR-126 inhibitor plusNIL (50 mg/kg, daily by gavage), and CpG-scrRNA plus NIL for 3 weeks.Percentage of donor CML cells in peripheral blood (PB) (FIG. 24A),spleen (FIG. 24B) and bone marrow (BM) (FIG. 24C), numbers of donor CMLLSK in spleen (FIG. 24D) and BM (FIG. 24E), and numbers of donor CMLlong term hematopoietic stem cells (LTHSC) in spleen (FIG. 24F) and BM(FIG. 24G) after 3 weeks' treatment were measured. Another cohort ofmice was treated for 3 weeks and then followed for survival studiesafter 3 weeks of treatment (n=10 in each group) (FIG. 24H). BM cells(CD45.2) from treated leukemic mice (3 weeks) were pooled, and 4×10⁶,2×10⁶, 1×10⁶, and 5×10⁵ cells/mouse were transplanted into secondarycongenic CD45.1 recipient mice irradiated at 900cGy (n=6mice/dose/condition×4 doses×4 conditions=96 mice). The recipient micewere monitored for 16 weeks for CML cell engraftment in blood andleukemia development by WBC count. Frequency of LIC was quantified usingL-Calc software (FIG. 24I). Abbreviations: NIL (Nilotinib); PB(peripheral blood); BM (bone marrow); LTHSC (long term hematopoieticstem cells); LIC (leukemia-initiating cells); LSK (lineage: Sca-1+c-kit+cells).

Other Embodiments

It is to be understood that while the disclosure has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of thedisclosure, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

Embodiments disclosed herein include embodiments P1 to P51 following.

Embodiment P1. An isolated compound comprising a phosphorothioatedoligodeoxynucleotide (ODN) conjugated to an anti-microRNA (anti-miR)sequence.

Embodiment P2. The compound of embodiment P1, wherein said anti-miRsequence is an anti-miR126, anti-miR142, anti-miR155, anti-miR9,anti-miR10b, anti-miR21, anti-miR17, or anti-miR92 nucleic acidsequence.

Embodiment P3. An isolated compound comprising a phosphorothioatedoligodeoxynucleotide (ODN) conjugated to a microRNA (miRNA) mimicnucleic acid sequence (miRNA-mimic).

Embodiment P4. The compound of embodiment P3, wherein said mimic is amiR126-mimic, miR142-mimic, miR155-mimic, miR9-mimic, miR10b-mimic,miR21-mimic, miR17-mimic, or miR92-mimic nucleic acid sequence

Embodiment P5. The compound of either of embodiment P1 or embodiment P3,further comprising one or more linkers between the ODN and anti-miR ormiRNA-mimic sequence, respectively.

Embodiment P6. The compound of embodiment P5, wherein the linkercomprises a substituted or unsubstituted alkylene or heteroalkylenelinker.

Embodiment P 7. The compound of embodiment P6, wherein the substitutedalkylene or heteroalkylene linker comprises a moiety selected from thegroup consisting of: an azide group, a protected amino group,N-hydroxysuccinimide (NHS) group, and a protected sulfhydryl group.

Embodiment P8. The compound of embodiment P7, wherein the substitutedalkylene or heteroalkylene linker comprising a protected sulfhydrylgroup is conjugated to a moiety selected from the group consisting of:divinyl sulfone derivative, acryloyl derivative, and maleimidoderivative.

Embodiment P9. The compound of embodiment P8, wherein the acryloylderivative is acryloyl chloride.

Embodiment P10. The compound of embodiment P6, wherein the substitutedalkylene or heteroalkylene linker is conjugated to polyethylene glycol(PEG) or bisphosphonate moiety.

Embodiment P11. The compound of embodiment P6, wherein the alkylene orheteroalkylene linker comprises an unsubstituted C₃ heteroalkylene.

Embodiment P12. The compound of embodiment P6, wherein the alkylene orheteroalkylene linker comprises an unsubstituted C₆ to C₁₂heteroalkylene.

Embodiment P13. The compound of embodiment P5, wherein the linker is asubstituted or unsubstituted alkylene, substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene.

Embodiment P14. The compound of embodiment P5, wherein the linker is asubstituted or unsubstituted C₁-C₄₀ alkylene, substituted orunsubstituted 2 to 40 membered heteroalkylene, substituted orunsubstituted C₃-C₈ cycloalkylene, substituted or unsubstituted 3 to 8membered heterocycloalkylene, substituted or unsubstituted C₆-C₁₀arylene, or substituted or unsubstituted 5 to 10 membered heteroarylene.

Embodiment P15. The compound of embodiment P5, wherein the linker is anunsubstituted C₁-C₄₀ alkylene, unsubstituted 2 to 40 memberedheteroalkylene, unsubstituted C₃-C₈ cycloalkylene, unsubstituted 3 to 8membered heterocycloalkylene, unsubstituted C₆-C₁₀ arylene, orunsubstituted 5 to 10 membered heteroarylene.

Embodiment P16. The compound of embodiment P5, wherein the linker is asubstituted 2 to 40 membered heteroalkylene.

Embodiment P17. The compound of either of embodiment P1 or embodimentP3, wherein said anti-miR or miRNA-mimic sequence, respectively, ischemically modified.

Embodiment P18. The compound of embodiment P17, wherein said anti-miR ormiRNA mimic sequence, respectively, comprises a chemical modificationselected from the group consisting of a 2′ 0-Methyl, 2′-deoxy-2′fluoro,2′-deoxy, a universal base, 5-C-methyl, an inverted deoxy abasic residueincorporation, and a locked nucleic acid.

Embodiment P19. The compound of embodiment P18, wherein saidmodification is positioned at the terminal nucleobase of said anti-miRor miRNA-mimic sequence, respectively.

Embodiment P20. The compound of embodiment P18, wherein the modificationis not positioned at the terminal nucleobase of said anti-miR ormiRNA-mimic sequence, respectively.

Embodiment P21. The compound of embodiment P18, wherein saidmodification protects against serum-derived nucleases.

Embodiment P22. The compound of either of embodiment P1 or embodimentP3, wherein said ODN sequence comprises a CpG-ODN nucleic acid sequenceselected from the group consisting of: a Class A CpG-ODN nucleic acidsequence, a Class B CpG-ODN nucleic acid sequence, and a Class C CpG-ODNnucleic acid sequence.

Embodiment P23. The compound of embodiment P1 or P3, wherein said ODNcomprises phosphodiester derivative linkage.

Embodiment P24. The compound of embodiment P23, wherein saidphosphodiester derivative linkage in said CpG nucleic acid sequence isselected from the group consisting of: a phosphoramidate linkage,phosphorodiamidate linkage, phosphorothioate linkage, phosphorodithioatelinkage, phosphonocarboxylic acid linkage, phosphonocarboxylate linkage,phosphonoacetic acid linkage, phosphonoformic acid linkage, methylphosphonate linkage, boron phosphonate linkage, andO-methylphosphoroamidite linkage.

Embodiment P25. A pharmaceutical composition comprising apharmaceutically acceptable excipient and the compound of either ofembodiment P1 or embodiment P3.

Embodiment P26. The pharmaceutical composition of embodiment P25,further comprising a second therapeutic agent.

Embodiment P27. The pharmaceutical composition of embodiment P26,wherein the second therapeutic agent is selected from the groupconsisting of: anti-tumor or anti-cancer agent, cytotoxic agent,cytostatic agent, anti-inflammatory agent, analgesic, anti-infectiveagent, growth inhibitory agent, immunogenic agent, immunomodulatoryagent, and chemokine.

Embodiment P28. The pharmaceutical composition of embodiment P27,wherein said anti-cancer agent is a cell death promoting agent.

Embodiment P29. The pharmaceutical composition of embodiment P27,wherein said second therapeutic agent is selected from the groupconsisting of: Actinomycin D/Dactinomycin, Bleomycin, Daunorubicin,Doxorubicin, Doxorubicin (pegylated liposomal), Epirubicin, Idarubicin,Mitomycin, Mitoxantrone, Etoposide, Docetaxel, Irinotecan, Paclitaxel,Topotecan, Vinblastine, Vincristine, Vinorelbine, Carboplatin,Cisplantin, Oxaliplatin, Alemtuzamab, BCG, Bevacizumab, Cetuximab,Denosumab, Erlotinib, Gefitinib, Imatinib, Interferon, Ipilimumab,Lapatinib, Monomethyl auristatin E (MMEA), Mertansine (DM1), Rituximab,Sunitinib, Sorafenib, Temsirolimus, and Trastuzumab, or anycombination(s) thereof.

Embodiment P30. A method of treating cancer in a subject in needthereof, the method comprising administering to said subject aneffective amount of the compound of either of embodiment P1 orembodiment P3 or the pharmaceutical composition of embodiment P25.

Embodiment P31. The method of embodiment P30, wherein the compound ofeither of embodiment P1 or embodiment P3, or the pharmaceuticalcomposition of embodiment P25, comprises anti-miR126 sequence ormiR142-mimic, respectively.

Embodiment P32. The method of embodiment P30, wherein the cancer is ahematopoietic cell cancer.

Embodiment P33. The method of embodiment P30, wherein the cancer in nota hematopoietic cell cancer.

Embodiment P34. The method of embodiment P30, wherein the cancer ismyeloma or acute myeloid leukemia.

Embodiment P35. The method of embodiment P30, wherein the cancer isprostate cancer, breast cancer, glioblastoma, ovarian cancer, lungcancer, head and neck cancer, esophageal cancer, skin cancer, melanoma,brain cancer, colorectal cancer, lymphoma, or myeloma, pancreaticcancer, chronic myeloid leukemia (CML), or myelodysplastic syndromes(MDS).

Embodiment P36. The method of any one of embodiments P30-P35, whereinthe compound or the composition is administered to the subject byintravenous, parenteral, subcutaneous, intramuscular, transdermal,intraperitoneal, intranasal, aerosol, oral, or topical administration.

Embodiment P37. The method of any one of embodiments P30-P36, whereinsaid treatment is dose-dependent of said compound or composition.

Embodiment P38. The method of any one of embodiments P30-P36, whereinabout 0.001 mg/kg to about 100 mg/kg of said compound is administered tosaid subject.

Embodiment P39. The method of any one of embodiments P30-P35, whereinsaid cancer is a relapsed cancer after chemotherapy.

Embodiment P40. The method of embodiment P39, wherein the relapsedcancer is chemotherapy resistant.

Embodiment P41. The method of any one of embodiments P30-P40, whereinsaid compound or said composition promotes cell-cycle entry of cancerstem cells, thereby treating said cancer.

Embodiment P42. The method of embodiment P41, wherein said cancer stemcells are leukemic stem cells (LSCs).

Embodiment P43. The method of embodiment P42, wherein said LSCs areCD34⁺CD38+ committed progenitor cells or primitive CD34⁺CD38− progenitorcells.

Embodiment P44. A method of reducing the activity of microRNA in a cellcomprising contacting the cell with an effective amount of the compoundof embodiment P1.

Embodiment P45. The method of one of embodiment P44, wherein said cellis a cancer cell.

Embodiment P46. The method of embodiment P45, wherein said cell is anacute myeloid lymphoid (AML) cell, prostate cancer cell, breast cancercell, glioblastoma cell, ovarian cancer cell, lung cancer cell, head andneck cancer cell, esophageal cancer cell, skin cancer cell, melanomacell, brain cancer cell, colorectal cancer cell, lymphoma cell, myelomacell, pancreatic cancer cell, chronic myeloid leukemia (CML cell, ormyelodysplastic syndromes (MDS) cell.

Embodiment P47. The method of embodiment P46, wherein said AML cell isfrom the bone marrow.

Embodiment P48. The method of any one of embodiments P44-P47, whereinsaid cell is a cultured cell in vitro.

Embodiment P49. The method of any one of embodiments P44-P47, whereinsaid cell is in situ in a host.

Embodiment P50. The method of any one of embodiments P44-P47, whereinsaid cell is in a cultured tissue ex vivo.

Embodiment P51. The method of any one of embodiments P44-P47, whereinsaid contacting step is free of viral transduction.

Embodiment P52. The method of any one of embodiments P44-P47, whereinsaid contacting step is free of viral transduction and said cell iscontacted with the compound of embodiment P1.

Embodiment P53. The method of any one of embodiments P44-P52, whereinsaid cell is contacted with about 1-100 nanomolar concentration of saidcompound.

What is claimed is:
 1. A compound comprising a phosphorothioated CpGoligodeoxynucleotide (CpG-ODN) conjugated to a microRNA (miRNA) mimicnucleic acid sequence (miRNA mimic) or an anti-microRNA (anti-miR)nucleic acid sequence; wherein the miRNA mimic nucleic acid sequence isa miR126-mimic nucleic acid sequence, miR142-mimic nucleic acidsequence, miR155-mimic nucleic acid sequence, miR9-mimic nucleic acidsequence, miR10b-mimic nucleic acid sequence, miR21-mimic nucleic acidsequence, miR17-mimic nucleic acid sequence, miR92-mimic nucleic acidsequence, miR125b-mimic nucleic acid sequence or miR146a-mimic nucleicacid sequence; and wherein the anti-miR nucleic acid sequence is ananti-miR126 nucleic acid sequence, anti-miR142 nucleic acid sequence,anti-miR155 nucleic acid sequence, anti-miR9 nucleic acid sequence,anti-miR10b nucleic acid sequence, anti-miR21 nucleic acid sequence,anti-miR17 nucleic acid sequence, anti-miR92 nucleic acid sequence,anti-miR125b nucleic acid sequence or anti-miR146a nucleic acidsequence.
 2. The compound of claim 1, further comprising a covalentlinker between the CpG-ODN and anti-miR nucleic acid sequence ormiRNA-mimic nucleic acid sequence.
 3. The compound of claim 2, whereinthe covalent linker is substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene.
 4. The compound of claim 2,wherein the covalent linker is unsubstituted C₁-C₄₀ alkylene,unsubstituted 2 to 40 membered heteroalkylene, unsubstituted C₃-C₈cycloalkylene, unsubstituted 3 to 8 membered heterocycloalkylene,unsubstituted C₆-C₁₀ arylene, or unsubstituted 5 to 10 memberedheteroarylene.
 5. The compound of claim 2, wherein the covalent linkeris substituted 2 to 40 membered heteroalkylene.
 6. The compound of claim1, wherein the anti-miR nucleic acid sequence or miRNA-mimic nucleicacid sequence is chemically modified.
 7. The compound of claim 6,wherein the anti-miR nucleic acid sequence or miRNA mimic nucleic acidsequence comprises a chemical modification selected for the groupconsisting of a 2′ 0-Methyl, 2′-deoxy-2′fluoro, 2′-deoxy, a universalbase, 5-C-methyl, an inverted deoxy abasic residue incorporation, and alocked nucleic acid.
 8. The compound of claim 7, wherein themodification is positioned at the terminal nucleobase of the anti-miRnucleic acid sequence or miRNA-mimic nucleic acid sequence.
 9. Thecompound of claim 1, wherein the CpG-ODN nucleic acid sequence isselected from the group consisting of a Class A CpG-ODN nucleic acidsequence, a Class B CpG-ODN nucleic acid sequence, and a Class C CpG-ODNnucleic acid sequence.
 10. The compound of claim 1, wherein the CpG-ODNcomprises phosphodiester derivative linkage selected from the groupconsisting of a phosphoramidate linkage, phosphorodiamidate linkage,phosphorodithioate linkage, phosphonocarboxylic acid linkage,phosphonocarboxylate linkage, phosphonoacetic acid linkage,phosphonoformic acid linkage, methyl phosphonate linkage, boronphosphonate linkage, and O-methylphosphoroamidite linkage.
 11. Thecompound of claim 1, wherein the miRNA-126 mimic nucleic acid sequencecomprises the nucleic acid sequence of any one of SEQ ID NOS:18-21. 12.The compound of claim 1, wherein the miRNA-126 mimic nucleic acidsequence has at least 80% sequence identity to SEQ ID NO:37.
 13. Thecompound of claim 1, wherein the miRNA-126 mimic nucleic acid sequenceis a double stranded oligomer of 20 to 30 bases.
 14. The compound ofclaim 1, wherein the phosphorothioated CpG oligodeoxynucleotidecomprises the nucleic acid sequence of any one of SEQ ID NOS:1-14, andthe miRNA-126 mimic nucleic acid sequence comprises the nucleic acidsequence of any one of SEQ ID NOS:18-21.
 15. A pharmaceuticalcomposition comprising the compound of claim 1 and a pharmaceuticallyacceptable excipient.
 16. The pharmaceutical composition of claim 15,further comprising a second therapeutic agent.
 17. A method of treatinga cancer, an infectious disease, or an autoimmune disease in a subjectin need thereof, the method comprising administering to the subject aneffective amount of the compound of claim
 1. 18. The method of claim 17,further comprising administering to the subject an effective amount of asecond therapeutic agent.
 19. The method of claim 18, further comprisingadministering to the subject an effective amount of nilotinib, cytosinearabinoside, doxorubicin, dactinomycin, bleomycin, daunorubicin,epirubicin, idarubicin, mitomycin, mitoxantrone, etoposide, docetaxel,irinotecan, paclitaxel, topotecan, vinblastine, vincristine,vinorelbine, carboplatin, cisplatin, oxaliplatin, alemtuzumab, BacillusCalmette-Guérin, bevacizumab, cetuximab, denosumab, erlotinib,gefitinib, imatinib, interferon, ipilimumab, lapatinib, monomethylauristatin E, mertansine, rituximab, sunitinib, sorafenib, temsirolimus,trastuzumab, or a combination of two or more thereof.
 20. A compoundcomprising a phosphorothioated CpG oligodeoxynucleotide conjugated to ananti-microRNA126 nucleic acid sequence; wherein the phosphorothioatedCpG oligodeoxynucleotide comprises the nucleic acid sequence of any oneof SEQ ID NOS:1-14, and the anti-microRNA126 nucleic acid sequencecomprises the nucleic acid sequence of any one of SEQ ID NOS:16, 17,22-31, 33-36, and 48.